Insulin resistance improving agents

ABSTRACT

A compound or its salt inhibiting the activity of a protein having an amino acid sequence which is the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, an extracellular domain of the protein above, an antibody, an antisense polynucleotide, etc. are usable as insulin-sensitizing agents and preventives and/or remedies for diabetes, obesity, hyperlipemia, arteriosclerosis, hypertension, heart disease, and so on.

TECHNICAL FIELD

The present invention relates to agents for sensitizing insulin,diagnostic agents therefor, agents for the prevention and/or treatmentof diabetes and diagnostic agents therefor, as well as screeningthereof, and so on.

BACKGROND ART

Insulin sensitization is a pathological condition that insulinsensitivity decreases in tissues and especially in type II diabetes, isa major cause associated with the development or progress of diabetes,in addition to impaired insulin secretion. In general, many patientswith diabetes accompanying obesity have insulin sensitization. Thus,insulin sensitization is considered to be deeply involved in obesity. Itis further known that insulin sensitization is observed not only indiabetes but also in disorders caused by abnormalities in lipidmetabolism such as arteriosclerosis, etc. (Saltiel, A. R., Cell, 104,517-529, 2001).

While the mechanism of insulin sensitization largely remains unknownyet, a suggestion, as one point of view, is made on a similarity to themechanism of inflammation in animal test or on an experimental levelusing a culture cell system. For example, it is reported that whenlipopolysaccharide (LPS) was administered to pregnant rats, theoffspring were found to develop obesity and insulin sensitization atadult age (Nilsson et al., Endocrinol., 142, 2622-2630, 2001); TNF-α,which is one of inflammatory cytokines, is produced and/or secreted morein obese adipocytes to inhibit the action of insulin (Hotamisligil G. S.et al., Science, 259, 87-91, 1993); moreover, thiazolidine derivatives,which are insulin-sensitizing agents, have an anti-inflammatory action(Pasceri, V. et al., Circulation, 101, 235-238, 2000) and so on.Furthermore, fluctuation in expression of several proteins associatedwith inflammatory reactions, including LPS-binding protein are notedalso in ob/ob mice as obesity model mice (Soukas, A. et al., GenesDevelop, 14, 963-980, 2000).

Triggering receptor expressed on myeloid cells 2 (TREM-2) is a membraneprotein of one transmembrane type belonging to the immunoglobulinsuperfamily found to be a homolog protein of TREM-1 thought to beinvolved in inflammatory reactions (Bauchon, A. et al., J. Immunol.,164, 4991-4995, 2000, Daws M. R. et al., Eur. J. Immunol., 31, 783-791,2001). TREM-1 is abundantly expressed on neutrophils and on monocytes.It is known that its expression results in amplifying inflammation bypromoting the secretion of lipopolysaccharide-induced TNF-α orinterleukin-1β and by inhibiting the expression, acute inflammatoryresponse can be suppressed in mice (Bauchon, A. et al., Nature, 410,1103-1107, 2001). It is reported that TREM-2 interacts with DAP12 as inTREM-1 to effect signal transduction (Daws M. R. et al., Eur. J.Immunol., 31, 783-791, 2001); TREM-2 induces the expression of CCchemokine receptor 7 (CCR7) in dendritic cells or macrophages to takepart in maturing of dendritic cells (Bouchon, A. et al., J. Exp. Med.,194, 1111-1122, 2001); and TREM-2 is associated with NO production (DawsM. R. et al., Eur. J. Immunol., 31, 783-791, 2001).

Safe and excellent pharmaceuticals for sensitizing insulin have beenearnestly awaited.

DISCLOSURE OF THE INVENTION

The present inventors made extensive investigations to solve theforegoing problems and as a result, found TREM-2 from the group ofgenes, which expression was enhanced in adipocytes of KKA^(y) mice as amodel animal with obesity and insulin sensitization (Nishimura, M., Exp.Animal, 18, 147-157, 1969). Based on this finding, the inventors madefurther studies and have thus come to accomplish the present invention.

That is, the present invention provides the following features.

(1) An insulin-sensitizing agent which comprises a compound or its saltthat inhibits the activity of a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO: 1, its partial peptide, or a salt thereof.

(2) An insulin-sensitizing agent which comprises a compound or its saltthat inhibits the expression of a gene for a protein containing the sameor substantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO: 1.

(3) An insulin-sensitizing agent which comprises a compound or its saltthat inhibits the expression of a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO: 1.

(4) The insulin-sensitizing agent according to (1) through (3), which isan agent for the prevention and/or treatment of diabetes, obesity,hyperlipemia, arteriosclerosis, hypertension or heart disease.

(4a) The insulin-sensitizing agent according to (1) through (3), whichis an agent for the prevention and/or treatment of diabetes.

(4b) The insulin-sensitizing agent according to (1) through (3), whichis an agent for the prevention and/or treatment of hyperlipemia.

(4c) The insulin-sensitizing agent according to (1) through (3), whichis an agent for the prevention and/or treatment of arteriosclerosis.

(5) An insulin-sensitizing agent which comprises an extracellular domainof a protein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1; or asalt thereof.

(6) The insulin-sensitizing agent according to (5), wherein theextracellular domain is a partial peptide containing the 14-167 aminoacid sequence in the amino acid sequence represented by SEQ ID NO: 1.

(7) An agent for the prevention and/or treatment of diabetes, obesity,hyperlipemia, arteriosclerosis, hypertension or heart disease whichcomprises an extracellular domain of a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO: 1; or a salt thereof.

(8) An antisense polynucleotide containing the entire or part of a basesequence complementary or substantially complementary to a base sequenceof a polynucleotide encoding a protein containing the same orsubstantially the same amino acid sequence represented by SEQ ID NO: 1or its partial peptide.

(9) An insulin-sensitizing agent which comprises the antisensepolynucleotide according to (8).

(10) An agent for the prevention and/or treatment of diabetes, obesity,hyperlipemia, arteriosclerosis, hypertension or heart disease, whichcomprises the antisense polynucleotide according to (8).

(11) An insulin-sensitizing agent which comprises an antibody to aprotein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1, itspartial peptide, or a salt thereof.

(12) An agent for the prevention and/or treatment of diabetes, obesity,hyperlipemia, arteriosclerosis, hypertension or heart disease, whichcomprises an antibody to a protein containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO: 1, its partial peptide, or a salt thereof.

(13) A diagnostic agent for insulin sensitization, which comprises anantibody to a protein containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ ID NO:1, its partial peptide, or a salt thereof.

(14) A diagnostic agent for diabetes, obesity, hyperlipemia,arteriosclerosis, hypertension or heart disease, which comprises anantibody to a protein containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ ID NO:1, its partial peptide, or a salt thereof.

(15) A diagnostic agent for insulin sensitization, which comprises apolynucleotide encoding a protein containing the same or substantiallythe same amino acid sequence represented by SEQ ID NO: 1, or its partialpeptide.

(16) A diagnostic agent for diabetes, obesity, hyperlipemia,arteriosclerosis, hypertension or heart disease, which comprises apolynucleotide encoding a protein containing the same or substantiallythe same amino acid sequence represented by SEQ ID NO: 1, or its partialpeptide.

(17) A method of screening an insulin-sensitizing agent, which comprisesusing a protein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1, itspartial peptide, or a salt thereof.

(18) A method of screening an antagonist of a protein containing thesame or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO: 1, or a salt of the protein, whichcomprises using (i) the protein, its partial peptide, or a salt thereofand (ii) a ligand to the protein, its partial peptide, or a saltthereof.

(19) A method of screening an insulin-sensitizing agent, which comprisesusing (i) a protein containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO: 1,its partial peptide, or a salt thereof and (ii) a ligand to the protein,its partial peptide, or a salt thereof.

(20) A kit for screening an insulin-sensitizing agent, which comprises aprotein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1, itspartial peptide, or a salt thereof.

(21) A method of screening an insulin-sensitizing agent, which comprisesusing a polynucleotide encoding a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO: 1, or its partial peptide.

(22) A kit for screening an insulin-sensitizing agent which comprises apolynucleotide encoding a protein containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO: 1, or its partial peptide.

(23) An insulin-sensitizing agent, which is obtainable using thescreening method according to (17), (19) or (21), or the screening kitaccording to (20) or (22).

(24) The screening method according to (17), (18), (19) or (21), whereinsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO: 1 is the amino acid sequence represented bySEQ ID NO: 2.

(24a) The screening kit according to (20) or (22), wherein substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO: 1 is the amino acid sequence represented by SEQ ID NO: 2.

(25) A method of screening an agent for the prevention and/or treatmentof diabetes, obesity, hyperlipemia, arteriosclerosis, hypertension orheart disease, which comprises using a protein containing same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO: 1, its partial peptide, or a salt thereof.

(25a) A method of screening an agent for the prevention and/or treatmentof diabetes, obesity, hyperlipemia, arteriosclerosis, hypertension orheart disease, which comprises using (i) a protein containing same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO: 1, its partial peptide, or a salt thereof, and(ii) a ligand to the protein, its partial peptide, or a salt thereof.

(26) A kit for screening an agent for the prevention and/or treatment ofdiabetes, obesity, hyperlipemia, arteriosclerosis, hypertension or heartdisease, which comprises a protein containing same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO: 1, its partial peptide, or a salt thereof.

(27) A method of screening an agent for the prevention and/or treatmentof diabetes, obesity, hyperlipemia, arteriosclerosis, hypertension orheart disease, which comprises using a polynucleotide encoding a proteincontaining same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO: 1, or its partial peptide.

(28) A kit for screening an agent for the prevention and/or treatment ofdiabetes, obesity, hyperlipemia, arteriosclerosis, hypertension or heartdisease, which comprises a polynucleotide encoding a protein containingsame or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO: 1, or its partial peptide.

(29) An agent for the prevention and/or treatment of diabetes, obesity,hyperlipemia, arteriosclerosis, hypertension or heart disease, which isobtainable using the screening method according to (25) or (27), or thescreening kit according to (26) or (28).

(29a) An agent for the prevention and/or treatment of diabetes, which isobtainable using the screening method according to (25) or (27), or thescreening kit according to (26) or (28).

(29b) The screening method according to (25) or (27), whereinsubstantially the same amino acid sequence represented by SEQ ID NO: 1is the amino acid sequence represented by SEQ ID NO: 2.

(29c) The screening kit according to (26) or (28), wherein substantiallythe same amino acid sequence represented by SEQ ID NO: 1 is the aminoacid sequence represented by SEQ ID NO: 2.

(30) A method of sensitizing insulin, which comprises administering to amammal an effective dose of a compound or its salt that inhibits theactivity of a protein containing same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO: 1,its partial peptide, or a salt of the protein, or a compound or its saltthat inhibits the expression of a gene for the protein.

(31) A method of preventing and/or treating diabetes, which comprisesadministering to a mammal an effective dose of a compound or its saltthat inhibits the activity of a protein containing same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO: 1, its partial peptide, or a salt of the protein, or acompound or its salt that inhibits the expression of a gene for theprotein.

(32) Use of a compound or its salt that inhibits the activity of aprotein containing same or substantially the same amino acid sequence asthe amino acid sequence represented by SEQ ID NO: 1, its partialpeptide, or a salt of the protein, or a compound or its salt thatinhibits the expression of a gene for the protein, to manufacture aninsulin-sensitizing agent.

(33) Use of a compound or its salt that inhibits the activity of aprotein containing same or substantially the same amino acid sequence asthe amino acid sequence represented by SEQ ID NO: 1, its partialpeptide, or a salt of the protein, or a compound or its salt thatinhibits the expression of a gene for the protein, to manufacture anagent for the prevention and/or treatment of diabetes.

BEST MODE FOR CARRYING OUT THE INVENTION

The protein containing same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1(hereinafter sometimes referred to as the protein or the presentinvention or the protein used in the present invention) may be anyprotein derived from any cell of human and warm-blooded animals (e.g.,guinea pigs, rats, mice, rabbits, swine, sheep, bovine, monkey, etc.);any cell (e.g., hepatocytes, splenocytes, nerve cells, glial cells, βcells of pancreas, bone marrow cells, mesangial cells, Langerhans'cells, epidermic cells, epithelial cells, goblet cells, endothelialcells, smooth muscle cells, fibroblasts, fibrocytes, myocytes,adipocytes, immune cells (e.g., macrophages, T cells, B cells, naturalkiller cells, mast cells, neutrophils, basophils, eosinophils,monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells,osteoblasts, osteoclasts, mammary gland cells, hepatocytes orinterstitial cells; or the corresponding precursor cells, stem cells,cancer cells, etc.); or any tissues where such cells are present, suchas brain or any of brain regions (e.g., olfactory bulb, amygdaloidnucleus, basal ganglia, hippocampus, thalamus, hypothalamus, cerebralcortex, medulla oblongata, cerebellum), spinal cord, hypophysis,stomach, pancreas, kidney, liver, gonad, thyroid, gall-bladder, bonemarrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (e.g.,large intestine and small intestine), blood vessel, heart, thymus,spleen, submandibular gland, peripheral blood, prostate, testicle,testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc.; theproteins may also be synthetic proteins.

The amino acid sequence which has substantially the same amino acidsequence as that represented by SEQ ID NO: 1 includes an amino acidsequence having at least about 60% homology, preferably at least about70% homology, more preferably at least about 80% homology, much morepreferably at least about 90% homology and most preferably at leastabout 95% homology, to the amino acid sequence represented by SEQ ID NO:1.

As the protein which contains substantially the same amino acid sequenceas that represented by SEQ ID NO: 1, for example, a protein havingsubstantially the same amino acid sequence as that represented by SEQ IDNO: 1 and preferably having the activity substantially equivalent to theamino acid sequence represented by SEQ ID NO: 1, etc. are preferred.Examples of the protein which contains substantially the same amino acidsequence as that represented by SEQ ID NO: 1 include a proteincontaining the amino acid sequence represented by SEQ ID NO: 2, and thelike.

Examples of the substantially equivalent activity described aboveinclude a signal transduction activity [e.g., the intracellular signaltransduction activity of the protein of the present invention(preferably TREM-2), etc.], a ligand binding activity [e.g., the ligandbinding activity of the protein of the present invention (preferablyTREM-2) to a ligand or a low molecular substance; etc.] and the like.The term substantially equivalent is used to mean that the activitiesare the same in nature (e.g., physiologically or pharmacologically).Therefore, although it is preferred that activities such as the signaltransduction and ligand binding activities, etc. be equivalent (e.g.,about 0.01- to about 100-fold, preferably about 0.1- to about 10-fold,more preferably about 0.5- to about 2-fold), quantitative factors suchas a level of these activities, a molecular weight of the protein, etc.may be different.

The signal transduction and ligand binding activities can be determinedaccording to publicly known methods, e.g., the method described in J.Exp. Med., 194, 1111-1122, 2001, or methods with some modificationsthereof.

Signal of the protein of the present invention (preferably TREM-2)causes phosphorylation on, e.g., TREM-2, activates ERK (extracellularsignal-related protein) and promotes secretion of inflammatory cytokine(e.g., TNF-α, etc.). Thus, the signal transduction activity describedabove is determined by adding to, e.g., cells expressed with the proteinof the present invention (e.g., TREM-2-expressed animal cells), (a) aligand-containing fluid such as a microorganism lysate, a microorganismsupernatant, an eukaryotic cell lysate, an eukaryotic cell supernatant,etc., (b) a ligand itself, (c) a substance having a binding activity tothe protein of the present invention equivalent to a naturally occurringligand, or (d) an antibody activating the protein of the presentinvention (e.g., TREM-2), if necessary, and assaying (1) the level ofphosphorylated ERK produced, (2) the level of TNF-α produced andsecreted extracellularly or (3) the level of phosphorylated TREM-2produced.

The level of phosphorylated ERK or TNF-α produced can be assayed bypublicly known methods (e.g., western blotting, EIA, etc.) using ananti-phosphorylated ERK antibody or an anti-TNF-α antibody. The level ofphosphorylated TREM-2 produced can be assayed by publicly known methods(e.g., immunoprecipitation, western blotting, etc.) using an anti-TREM-2antibody and an anti-phosphorylated tyrosine antibody.

The ligand binding activity can be assayed by, e.g.,immunoprecipitation, protein affinity purification, yeast two-hybridtechniques, etc., using the protein of the present invention (preferablyTREM-2) and a ligand.

Examples of the protein used in the present invention include (1)so-called muteins such as proteins containing (i) the amino acidsequence represented by SEQ ID NO: 1, of which at least 1 or 2 (e.g.,about 1 to about 100, preferably about 1 to about 30, more preferablyabout 1 to about 10 and most preferably several (1 to 5)) amino acidsare deleted; (ii) the amino acid sequence represented by SEQ ID NO: 1,to which at least 1 or 2 (e.g., about 1 to about 100, preferably about 1to about 30, more preferably about 1 to about 10 and most preferablyseveral (1 to 5)) amino acids are added; (iii) the amino acid sequencerepresented by SEQ ID NO: 1, in which at least 1 or 2 (e.g., about 1 toabout 100, preferably about 1 to about 30, more preferably about 1 toabout 10 and most preferably several (1 to 5)) amino acids are inserted;(iv) the amino acid sequence represented by SEQ ID NO: 1, in which atleast 1 or 2 (e.g., about 1 to about 100, preferably about 1 to about30, more preferably about 1 to about 10 and most preferably several (1to 5)) amino acids are substituted by other amino acids; or (v) acombination of these amino acid sequences; and, (2) so-called muteinssuch as proteins containing (i) the amino acid sequence represented bySEQ ID NO: 2, of which at least 1 or 2 (e.g., about 1 to about 100,preferably about 1 to about 30, more preferably about 1 to about 10 andmost preferably several (1 to 5)) amino acids are deleted; (ii) theamino acid sequence represented by SEQ ID NO: 2, to which at least 1 or2 (e.g., about 1 to about 100, preferably about 1 to about 30, morepreferably about 1 to about 10 and most preferably several (1 to 5))amino acids are added; (iii) the amino acid sequence represented by SEQID NO: 2, in which at least 1 or 2 (e.g., about 1 to about 100,preferably about 1 to about 30, more preferably about 1 to about 10 andmost preferably several (1 to 5)) amino acids are inserted; (iv) theamino acid sequence represented by SEQ ID NO: 2, in which at least 1 or2 (e.g., about 1 to about 100, preferably about 1 to about 30, morepreferably about 1 to about 10 and most preferably several (1 to 5))amino acids are substituted by other amino acids; or (v) a combinationof these amino acid sequences; etc.

Where an amino acid sequence(s) are inserted, deleted or substituted asdescribed above, the positions of such insertion, deletion orsubstitution are not particularly limited.

Throughout the specification, the proteins are represented in accordancewith the conventional way of describing proteins, that is, theN-terminus (amino terminus) at the left hand and the C-terminus(carboxyl terminus) at the right hand. In the protein of the presentinvention including the protein containing the amino acid sequencerepresented by SEQ ID NO: 1, the C-terminus may be in any form of acarboxyl group (—COOH), a carboxylate (—COO⁻), an amide (—CONH₂) and anester (—COOR).

Herein, examples of the ester group shown by R include a C₁₋₆ alkylgroup such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc.; a C₃₋₈cycloalkyl group such as cyclopentyl, cyclohexyl, etc.; a C₆₋₁₂ arylgroup such as phenyl, α-naphthyl, etc.; a C₇₋₁₄ aralkyl such as aphenyl-C₁₋₂ alkyl group, e.g., benzyl, phenethyl, etc.; anα-naphthyl-C₁₋₂ alkyl group such as α-naphthylmethyl, etc.;pivaloyloxymethyl and the like.

Where the protein used in the present invention contains a carboxylgroup (or a carboxylate) at a position other than the C-terminus, thecarboxyl group may be amidated or esterified and such an amide or esteris also included within the protein used in the present invention.Examples of the ester group in this case may be the C-terminal estersdescribed above, etc.

Furthermore, examples of the protein used in the present inventioninclude variants wherein the amino group at the N-terminal amino acidresidues (e.g., methionine residue) is protected with a protecting group(e.g., a C₁₋₆ acyl group such as a C₁₋₆ alkanoyl group, e.g., formylgroup, acetyl group, etc.); those wherein the N-terminal region iscleaved in vivo and the glutamyl group thus formed is pyroglutaminated;those wherein a substituent (e.g., —OH, —SH, amino group, imidazolegroup, indole group, guanidino group, etc.) on the side chain of anamino acid in the molecule is protected with a suitable protecting group(e.g., a C₁₋₆ acyl group such as a C₁₋₆alkanoyl group, e.g., formylgroup, acetyl group, etc.), or conjugated proteins such as glycoproteinshaving sugar chains; etc.

Specific examples of the protein used in the present invention are aprotein containing the amino acid sequence represented by SEQ ID NO: 1(human TREM-2), a protein containing the amino acid sequence representedby SEQ ID NO: 2 (mouse TREM-2), and the like.

The partial peptide of the protein used in the present invention may beany peptide as long as it is a partial peptide of the protein used inthe present invention described above and preferably has the propertyequivalent to that of the protein used in the present inventiondescribed above.

Specifically, for the purpose of preparing the antibody of the presentinvention later described, there are a peptide having the 133-147 aminoacid sequence in the amino acid sequence represented by SEQ ID NO: 1, apeptide having the 133-147 amino acid sequence in the amino acidsequence represented by SEQ ID NO: 2, etc. Preferably used are peptidescontaining the sequence of, e.g., at least 20, preferably at least 50,more preferably at least 70, much more preferably at least 100, and mostpreferably at least 200, amino acids in the constituent amino acidsequence of the protein used the present invention, and the like.

The partial peptides used in the present invention may be peptidescontaining the amino acid sequence, of which at least 1 or 2 (preferablyabout 1 to about 10 and more preferably several (1 to 5)) amino acidsmay be deleted; peptides, to which at least 1 or 2 (preferably about 1to about 20, more preferably about 1 to about 10 and most preferablyseveral (1 to 5)) amino acids may be added; peptides, in which at least1 or 2 (preferably about 1 to about 20, more preferably about 1 to about10 and most preferably several (1 to 5)) amino acids may be inserted; orpeptides, in which at least 1 or 2 (preferably about 1 to about 10, morepreferably several and most preferably about 1 to about 5) amino acidsmay be substituted by other amino acids.

The partial peptides used in the present invention further includeextracellular domains of the protein used in the present invention. Theextracellular domains are, for example, a peptide having the 14-167 or19-173 amino acid sequence in the amino acid sequence represented by SEQID NO: 1, a peptide having the 14-170 or 19-170 amino acid sequence inthe amino acid sequence represented by SEQ ID NO: 2, and the like.

In the partial peptide of the present invention, the C-terminus may bein any form of a carboxyl group (—COOH), a carboxylate (—COO⁻), an amide(—CONH₂) or an ester (—COOR).

Furthermore, the partial peptide used in the present invention includesvariants having a carboxyl group (or a carboxylate) at a position otherthan the C-terminus, those wherein the amino group at the N-terminalamino acid residues (e.g., methionine residue) is protected with aprotecting group; those wherein the N-terminal region is cleaved in vivoand the glutamyl group thus formed is pyroglutaminated; those wherein asubstituent on the side chain of an amino acid in the molecule isprotected with a suitable protecting group, or conjugated proteins suchas so-called glycoproteins having sugar chains; etc., as in the proteinof the present invention described above.

The partial peptide used in the present invention may also be used as anantigen for producing antibodies.

As salts of the protein or partial peptide used in the presentinvention, salts with physiologically acceptable acids (e.g., inorganicacids or organic acids) or bases (e.g., alkali metal salts) may beemployed, preferably in the form of physiologically acceptable acidaddition salts. Examples of such salts include salts with inorganicacids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, andsulfuric acid), salts with organic acids (e.g., acetic acid, formicacid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaricacid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid, benzenesulfonic acid) and the like.

The protein or partial peptide used in the present invention or saltsthereof may be manufactured by publicly known methods of purifyingproteins from human or other warm-blooded animal cells or tissuesdescribed above. Alternatively, they may also be manufactured byculturing transformants containing DNAs encoding these proteins.Furthermore, they may also be manufactured by a modification of themethods for peptide synthesis, which will be described hereinafter.

Where these proteins are manufactured from human or mammalian tissues orcells, human or mammalian tissues or cells are homogenized, extractedwith an acid, etc., and the extract can be isolated and purified by acombination of chromatography techniques such as reverse phasechromatography, ion exchange chromatography, and the like.

To synthesize the protein or partial peptide used in the presentinvention or its salts, or amides thereof, commercially available resinsfor protein synthesis may be used. Examples of such resins includechloromethyl resin, hydroxymethyl resin, benzhydrylamine resin,aminomethyl resin, 4-benzyloxybenzyl alcohol resin,4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin,4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl) phenoxy resin, etc. Usingthese resins, amino acids, in which α-amino groups and functional groupson the side chains are appropriately protected, are condensed on theresin in the order of the sequence of the objective protein according tovarious condensation methods publicly known in the art. At the end ofthe reaction, the protein or partial peptide is excised from the resinand at the same time, the protecting groups are removed. Then,intramolecular disulfide bond-forming reaction is performed in a highlydiluted solution to obtain the objective protein or partial peptide, oramides thereof.

For condensation of the protected amino acids described above, a varietyof activation reagents for protein synthesis may be used, andcarbodiimides are particularly preferred. Examples of such carbodiimidesinclude DCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide, etc. For activation bythese reagents, the protected amino acids in combination with aracemization inhibitor (e.g., HOBt, HOOBt) are added directly to theresin, or the protected amino acids are previously activated in the formof symmetric acid anhydrides, HOBt esters or HOOBt esters, followed byadding the thus activated protected amino acids to the resin.

Solvents suitable for use to activate the protected amino acids orcondense with the resin may be chosen from solvents that are known to beusable for protein condensation reactions. Examples of such solvents areacid amides such as N,N-dimethylformamide, N,N-dimethylacetamide,N-methylpyrrolidone, etc.; halogenated hydrocarbons such as methylenechloride, chloroform, etc.; alcohols such as trifluoroethanol, etc.;sulfoxides such as dimethylsulfoxide, etc.; ethers such as pyridine,dioxane, tetrahydrofuran, etc.; nitriles such as acetonitrile,propionitrile, etc.; esters such as methyl acetate, ethyl acetate, etc.;and appropriate mixtures of these solvents. The reaction temperature isappropriately chosen from the range known to be applicable to proteinbinding reactions and is usually selected in the range of approximately−20° C. to 50° C. The activated amino acid derivatives are usedgenerally in an excess of 1.5 to 4 times. The condensation is examinedusing the ninhydrin reaction; when the condensation is insufficient, thecondensation can be completed by repeating the condensation reactionwithout removal of the protecting groups. When the condensation is yetinsufficient even after repeating the reaction, unreacted amino acidsare acetylated with acetic anhydride or acetylimidazole to cancel anypossible adverse affect on the subsequent reaction.

Examples of the protecting groups used to protect the starting aminogroups include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl,trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulphenyl,diphenylphosphinothioyl, Fmoc, etc.

A carboxyl group can be protected by, e.g., alkyl esterification(linear, branched or cyclic alkyl esterification of, e.g., methyl,ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, 2-adamantyl, etc.), aralkyl esterification (e.g., benzylester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester,benzhydryl ester, etc.), phenacyl esterification, benzyloxycarbonylhydrazidation, t-butoxycarbonyl hydrazidation, trityl hydrazidation, orthe like.

The hydroxyl group of serine can be protected through, for example, itsesterification or etherification. Examples of groups appropriately usedfor the esterification include a lower (C₁₋₆) alkanoyl group, such asacetyl group, an aroyl group such as benzoyl group, and a group derivedfrom carbonic acid such as benzyloxycarbonyl group, ethoxycarbonylgroup, etc. Examples of a group appropriately used for theetherification include benzyl group, tetrahydropyranyl group, t-butylgroup, etc.

Examples of groups for protecting the phenolic hydroxyl group oftyrosine include Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z, t-butyl, etc.

Examples of groups used to protect the imidazole moiety of histidineinclude Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP,benzyloxymethyl, Bum, Boc, Trt, Fmoc, etc.

Examples of the activated carboxyl groups in the starting materialinclude the corresponding acid anhydrides, azides, activated esters[esters with alcohols (e.g., pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccimide, N-hydroxyphthalimide, HOBt)].

As the amino acids in which the amino groups are activated in thestarting material, the corresponding phosphoric amides are employed.

To eliminate (split off) the protecting groups, there are used catalyticreduction under hydrogen gas flow in the presence of a catalyst such asPd-black or Pd-carbon; an acid treatment with anhydrous hydrogenfluoride, methanesulfonic acid, trifluoromethanesulfonic acid,trifluoroacetic acid, or a mixture solution of these acids; a treatmentwith a base such as diisopropylethylamine, triethylamine, piperidine orpiperazine; reduction with sodium in liquid ammonia, etc. Theelimination of the protecting group by the acid treatment describedabove is carried out generally at a temperature of approximately −20° C.to 40° C. In the acid treatment, it is efficient to add a cationscavenger such as anisole, phenol, thioanisole, m-cresol, p-cresol,dimethylsulfide, 1,4-butanedithiol, 1,2-ethanedithiol, etc. Furthermore,2,4-dinitrophenyl group known as the protecting group for the imidazoleof histidine is removed by a treatment with thiophenol. Formyl groupused as the protecting group of the indole of tryptophan is eliminatedby the aforesaid acid treatment in the presence of 1,2-ethanedithiol,1,4-butanedithiol, etc. as well as by a treatment with an alkali such asa dilute sodium hydroxide solution, dilute ammonia, etc.

Protection of functional groups that should not be involved in thereaction of the starting materials, protecting groups, elimination ofthe protecting groups and activation of functional groups involved inthe reaction may be appropriately selected from publicly known groupsand publicly known means.

In another method for obtaining the amides of the desired protein orpartial peptide, for example, the α-carboxyl group of the carboxyterminal amino acid is first protected by amidation; the peptide(protein) chain is then extended from the amino group side to a desiredlength. Then, a protein or partial peptide, in which only the protectinggroup of the N-terminal α-amino group of the peptide chain has beeneliminated, and a protein or partial peptide, in which only theprotecting group of the C-terminal carboxyl group has been eliminatedare manufactured. The two proteins or peptides are condensed in amixture of the solvents described above. The details of the condensationreaction are the same as described above. After the protected protein orpeptide obtained by the condensation is purified, all the protectinggroups are eliminated by the method described above to give the desiredcrude protein or peptide. This crude protein or peptide is purified byvarious known purification means. Lyophilization of the major fractiongives the amide of the desired protein or peptide.

To prepare the esterified protein or peptide, for example, theα-carboxyl group of the carboxy terminal amino acid is condensed with adesired alcohol to prepare the amino acid ester, which is followed byprocedures similar to the preparation of the amidated protein or peptideabove to give the desired esterified protein or peptide.

The partial peptide used in the present invention or salts thereof canbe manufactured by publicly known methods for peptide synthesis, or bycleaving the protein used in the present invention with an appropriatepeptidase. For the methods for peptide synthesis, for example, eithersolid phase synthesis or liquid phase synthesis may be used. That is,the partial peptide or amino acids that can construct the partialpeptide used in the present invention are condensed with the remainingpart. Where the product contains protecting groups, these protectinggroups are removed to give the desired peptide. Publicly known methodsfor condensation and elimination of the protecting groups are describedin (i) to (v) below.

-   (i) M. Bodanszky & M. A. Ondetti: Peptide Synthesis, Interscience    Publishers, New York (1966)-   (ii) Schroeder & Luebke: The Peptide, Academic Press, New York    (1965)-   (iii) Nobuo Izumiya, et al.: Peptide Gosei-no-Kiso to Jikken (Basics    and experiments of peptide synthesis), published by Maruzen Co.    (1975)-   (iv) Haruaki Yajima & Shunpei Sakakibara: Seikagaku Jikken Koza    (Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry of    Proteins) IV, 205 (1977)-   (v) Haruaki Yajima ed.: Zoku lyakuhin no Kaihatsu (A sequel to    Development of Pharmaceuticals), Vol. 14, Peptide Synthesis,    published by Hirokawa Shoten

After completion of the reaction, the partial peptide used in thepresent invention may be purified and isolated by a combination ofconventional purification methods such as solvent extraction,distillation, column chromatography, liquid chromatography andrecrystallization to give the partial peptide of the present invention.When the partial peptide obtained by the above methods is in a freeform, the partial peptide can be converted into an appropriate salt by apublicly known method; when the partial peptide is obtained in a saltform, it can be converted into a free form or other different salt formby a publicly known method.

The polynucleotide encoding the protein used in the present inventionmay be any polynucleotide so long as it contains the base sequenceencoding the protein used in the present invention described above.Preferably, the polynucleotide is a DNA. The DNA may be any one ofgenomic DNA, genomic DNA library, cDNA derived from the cells and/ortissues described above, cDNA library derived from the cells and/ortissues described above and synthetic DNA.

The vector used for the library may be any of bacteriophage, plasmid,cosmid, phagemid and the like. In addition, the DNA can be amplified byreverse transcriptase polymerase chain reaction (hereinafter abbreviatedas RT-PCR) with total RNA or mRNA fraction prepared from theabove-described cells and/or tissues.

The DNA encoding the protein used in the present invention may be anyone of a DNA having, for example, the base sequence represented by SEQID NO: 3, or any DNA having a base sequence hybridizable to the basesequence represented by SEQ ID NO: 3 under high stringent conditions andencoding a protein which has the properties substantially equivalent tothose of the protein containing the amino acid sequence represented bySEQ ID NO: 1.

Specific examples of the DNA that is hybridizable to the base sequencerepresented by SEQ ID NO: 3 under high stringent conditions include DNAscontaining the base sequence having at least about 60% homology,preferably at least about 70% homology, preferably at least about 80%homology, preferably at least about 90% homology and preferably at leastabout 95% homology, to the base sequence represented by SEQ ID NO: 4;etc.

The hybridization can be carried out by publicly known methods or bymodifications thereof, for example, according to the method described inMolecular Cloning, 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab.Press, 1989). A commercially available library can also be usedaccording to the instructions of the attached manufacturer's protocol.The hybridization can be carried out preferably under high stringentconditions.

The high stringent conditions are, for example, those in a sodiumconcentration at about 19 to 40 mM, preferably about 19 to 20 mM at atemperature of about 50 to 70° C., preferably about 60 to 65° C. Inparticular, hybridization conditions in a sodium concentration of about19 mM at a temperature of about 65° C. are most preferred.

More specifically, as the DNA encoding the protein containing the aminoacid sequence represented by SEQ ID NO: 1, there may be employed a DNAcontaining the base sequence represented by SEQ ID NO: 3. As the DNAencoding the protein containing the amino acid sequence represented bySEQ ID NO: 2, there may be employed a DNA containing the base sequencerepresented by SEQ ID NO: 4, etc.

The DNA encoding the partial peptide used in the present invention maybe any DNA so long as it contains the base sequence encoding the partialpeptide used in the present invention described above. The DNA may alsobe any of genomic DNA, genomic DNA library, cDNA derived from the cellsand/or tissues described above, cDNA library derived from the cellsand/or tissues described above and synthetic DNA.

As the DNA encoding the partial peptide used in the present invention,there are employed, for example, a DNA containing a part of DNAcontaining the base sequence represented by SEQ ID NO: 3, or a DNAcontaining a base sequence hybridizable to the base sequence representedby SEQ ID NO: 3 under high stringent conditions and containing a part ofDNA encoding a protein having the activities substantially equivalent tothose of the protein of the present invention; etc.

The DNA hybridizable to the base sequence represented by SEQ ID NO: 3has the same significance as described above.

The DNA hybridizable to the base sequence represented by SEQ ID NO: 3has the same significance as described above.

Methods for the hybridization and the high stringent conditions that canbe used are the same as those described above.

For cloning of DNAs that completely encode the protein or partialpeptide used in the present invention (hereinafter sometimes merelyreferred to as the protein of the present invention in the descriptionof cloning of DNAs encoding the protein and partial peptide and theirexpression), the DNA can be either amplified by PCR using synthetic DNAprimers containing a part of the base sequence of the protein of thepresent invention, or the DNA inserted into an appropriate vector can bescreened by hybridization with a labeled DNA fragment or synthetic DNAthat encodes a part or entire region of the protein of the presentinvention. The hybridization can be carried out, for example, accordingto the method described in Molecular Cloning, 2nd (J. Sambrook et al.,Cold Spring Harbor Lab. Press, 1989). Where the hybridization is carriedout using commercially available library, the procedures may beconducted in accordance with the protocol described in the attachedinstructions.

Conversion of the base sequence of DNA can be effected by publicly knownmethods such as PCR, the ODA-LA PCR method, the Gapped duplex method,the Kunkel method, etc., or its modification, using a publicly known kitavailable as Mutan™-super Express Km (manufactured by Takara Shuzo Co.,Ltd.) or Mutan™-K (manufactured by Takara Shuzo Co., Ltd.), etc.

The cloned DNA encoding the protein can be used as it is, depending uponpurpose or, if desired, after digestion with a restriction enzyme orafter addition of a linker thereto. The DNA may contain ATG as atranslation initiation codon at the 5′ end thereof and TAA, TGA or TAGas a translation termination codon at the 3′ end thereof. Thesetranslation initiation and termination codons may also be added by usingan appropriate synthetic DNA adapter.

The expression vector for the protein of the present invention can bemanufactured, for example, by (a) excising the objective DNA fragmentfrom the DNA encoding the protein of the present invention, and then (b)ligating the DNA fragment with an appropriate expression vectordownstream a promoter in the vector.

Examples of the vector include plasmids derived form E. coli (e.g.,pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis(e.g., pUB110, pTP5, pC194), plasmids derived from yeast (e.g., pSH19,pSH15), bacteriophages such as λ phage, etc., animal viruses such asretrovirus, vaccinia virus, baculovirus, etc. as well as pA1-11, pXT1,pRc/CMV, pRc/RSV, pcDNA I/Neo, etc.

The promoter used in the present invention may be any promoter if itmatches well with a host to be used for gene expression. In the case ofusing animal cells as the host, examples of the promoter include SRαpromoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter,etc.

Among them, it is preferred to use CMV (cytomegalovirus) promoter, SRαpromoter, etc. Where the host is bacteria of the genus Escherichia,preferred examples of the promoter include trp promoter, lac promoter,recA promoter, λP_(L) promoter, lpp promoter, T7 promoter, etc. In thecase of using bacteria of the genus Bacillus as the host, preferredexample of the promoter are SPO1 promoter, SPO2 promoter, penP promoter,etc. When yeast is used as the host, preferred examples of the promoterare PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. Wheninsect cells are used as the host, preferred examples of the promoterinclude polyhedrin prompter, P10 promoter, etc.

In addition to the foregoing examples, the expression vector may furtheroptionally contain an enhancer, a splicing signal, a poly A additionsignal, a selection marker, SV40 replication origin (hereinaftersometimes abbreviated as SV40ori), etc. Examples of the selection markerinclude dihydrofolate reductase (hereinafter sometimes abbreviated asdhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene(hereinafter sometimes abbreviated as Amp^(r)), neomycin resistant gene(hereinafter sometimes abbreviated as Neo^(r), G418 resistance), etc. Inparticular, when dhfr gene is used as the selection marker using dhfrgene-deficient Chinese hamster cells, selection can also be made on athymidine free medium.

If necessary, a signal sequence that matches with a host is added to theN-terminus of the protein of the present invention. Examples of thesignal sequence that can be used are PhoA signal sequence, OmpA signalsequence, etc. when bacteria of the genus Escherichia is used as thehost; α-amylase signal sequence, subtilisin signal sequence, etc. whenbacteria of the genus Bacillus is used as the host; MFα signal sequence,SUC2 signal sequence, etc. when yeast is used as the host; and insulinsignal sequence, α-interferon signal sequence, antibody molecule signalsequence, etc. when animal cells are used as the host, respectively.

Using the vector containing the DNA encoding the protein of the presentinvention thus constructed, transformants can be manufactured.

Examples of the host, which may be employed, are bacteria belonging tothe genus Escherichia, bacteria belonging to the genus Bacillus, yeast,insect cells, insects and animal cells, etc.

Specific examples of the bacteria belonging to the genus Escherichiainclude Escherichia coli K12 DH1 [Proc. Natl. Acad. Sci. U.S.A., 60, 160(1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journalof Molecular Biology, 120, 517 (1978)], HB101 [Journal of MolecularBiology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], etc.

Examples of the bacteria belonging to the genus Bacillus includeBacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal ofBiochemistry, 95, 87 (1984)], etc.

Examples of yeast include Saccharomyces cereviseae AH22, AH22R⁻,NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036,Pichia pastoris KM71, etc.

Examples of insect cells include, for the virus AcNPV, Spodopterafrugiperda cell (Sf cell), MG1 cell derived from mid-intestine ofTrichoplusia ni, High Five™ cell derived from egg of Trichoplusia ni,cells derived from Mamestra brassicae, cells derived from Estigmenaacrea, etc.; and for the virus BmNPV, Bombyx mori N cell (BmN cell),etc. is used. Examples of the Sf cell which can be used are Sf9 cell(ATCC CRL1711), Sf21 cell (both cells are described in Vaughn, J. L. etal., In Vivo, 13, 213-217 (1977), etc.

As the insect, for example, a larva of Bombyx mori can be used [Maeda etal., Nature, 315, 592 (1985)].

Examples of animal cells include monkey cell COS-7, Vero, Chinesehamster cell CHO (hereinafter referred to as CHO cell), dhfrgene-deficient Chinese hamster cell CHO (hereinafter simply referred toas CHO (dhfr⁻) cell), mouse L cell, mouse AtT-20, mouse myeloma cell,mouse ATDC5 cell, rat GH3, human FL cell, 3T3-L1 cell, L6 cell, C2C12cell, etc.

Bacteria belonging to the genus Escherichia can be transformed, forexample, by the method described in Proc. Natl. Acad. Sci. U.S.A., 69,2110 (1972), Gene, 17, 107 (1982), etc.

Bacteria belonging to the genus Bacillus can be transformed, forexample, by the method described in Molecular & General Genetics, 168,111 (1979), etc.

Yeast can be transformed, for example, by the method described inMethods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci.U.S.A., 75, 1929 (1978), etc.

Insect cells or insects can be transformed, for example, according tothe method described in Bio/Technology, 6, 47-55 (1988), etc.

Animal cells can be transformed, for example, according to the methoddescribed in Saibo Kogaku (Cell Engineering), extra issue 8, Shin SaiboKogaku Jikken Protocol (New Cell Engineering Experimental Protocol),263-267 (1995) (published by Shujunsha), or Virology, 52, 456 (1973).

Thus, the transformants transformed with the expression vectorscontaining the DNAs encoding the protein can be obtained.

Where the host is bacteria belonging to the genus Escherichia or thegenus Bacillus, the transformant can be appropriately cultured in aliquid medium which contains materials required for growth of thetransformant such as carbon sources, nitrogen sources, inorganicmaterials, and the like. Examples of the carbon sources include glucose,dextrin, soluble starch, sucrose, etc.; examples of the nitrogen sourcesinclude inorganic or organic materials such as ammonium salts, nitratesalts, corn steep liquor, peptone, casein, meat extract, soybean cake,potato extract, etc.; and, examples of the inorganic materials arecalcium chloride, sodium dihydrogenphosphate, magnesium chloride, etc.In addition, yeast extracts, vitamins, growth promoting factors etc. mayalso be added to the medium. Preferably, pH of the medium is adjusted toabout 5 to about 8.

A preferred example of the medium for culturing the bacteria belongingto the genus Escherichia is M9 medium supplemented with glucose andCasamino acids [Miller, Journal of Experiments in Molecular Genetics,431-433, Cold Spring Harbor Laboratory, New York, 1972]. If necessary, achemical such as 3β-indolylacrylic acid can be added to the mediumthereby to activate the promoter efficiently.

Where the bacteria belonging to the genus Escherichia are used as thehost, the transformant is usually cultivated at about 15 to 43° C. forabout 3 to 24 hours. If necessary, the culture may be aerated oragitated.

Where the bacteria belonging to the genus Bacillus are used as the host,the transformant is cultured generally at about 30 to 40° C. for about 6to 24 hours. If necessary, the culture can be aerated or agitated.

Where yeast is used as the host, the transformant is cultivated, forexample, in Burkholder's minimal medium [Bostian, K. L. et al., Proc.Natl. Acad. Sci. U.S.A., 77, 4505 (1980)] or in SD medium supplementedwith 0.5% Casamino acids [Bitter, G A. et al., Proc. Natl. Acad. Sci.U.S.A., 81, 5330 (1984)]. Preferably, the pH of medium is adjusted toapproximately 5 to 8. In general, the transformant is cultivated atapproximately 20 to 35° C. for approximately 24 to 72 hours. Ifnecessary, the culture can be aerated or agitated.

Where insect cells or insects are used as the host, the transformant iscultivated in, for example, Grace's Insect Medium (Nature, 195, 788(1962)) to which an appropriate additive such as immobilized 10% bovineserum is added. Preferably, pH of the medium is adjusted to about 6.2 toabout 6.4. Normally, the transformant is cultivated at about 27° C. forabout 3 days to about 5 days and, if necessary, the culture can beaerated or agitated.

Where animal cells are employed as the host, the transformant iscultured in, for example, MEM medium containing about 5 to 20% fetalbovine serum [Science, 122, 501 (1952)], DMEM medium [Virology, 8, 396(1959)], RPMI 1640 medium [The Journal of the American MedicalAssociation, 199, 519 (1967)], 199 medium [Proceeding of the Society forthe Biological Medicine, 73, 1 (1950)], etc. Preferably, pH of themedium is adjusted to about 6 to about 8. The transformant is usuallycultivated at about 30° C. to about 40° C. for about 15 to about 60hours and, if necessary, the culture can be aerated or agitated.

As described above, the protein of the present invention can be producedin the transformant, in the cell membrane of the transformant, oroutside of the transformant.

The protein of the present invention can be separated and purified fromthe culture described above by the following procedures.

When the protein of the present invention is extracted from the bacteriaor cells, the bacteria or cell is collected after culturing by apublicly known method and suspended in an appropriate buffer. Thebacteria or cell is then disrupted by publicly known methods such asultrasonication, a treatment with lysozyme and/or freeze-thaw cycling,followed by centrifugation, filtration, etc. to give the crude extractof the protein. These procedures are appropriately used. The buffer usedfor the procedures may contain a protein modifier such as urea orguanidine hydrochloride, or a surfactant such as Triton X-100™, etc.When the protein of the present invention is secreted in the culturebroth, the supernatant can be separated, after completion of thecultivation, from the bacteria or cell to collect the supernatant by apublicly known method.

The protein contained in the supernatant or the extract thus obtainedcan be purified by appropriately combining the publicly known methodsfor separation and purification. Such publicly known methods forseparation and purification include a method utilizing difference insolubility such as salting out, solvent precipitation, etc.; a methodmainly utilizing difference in molecular weight such as dialysis,ultrafiltration, gel filtration, SDS-polyacrylamide gel electrophoresis,etc.; a method utilizing difference in electric charge such as ionexchange chromatography, etc.; a method utilizing difference in specificaffinity such as affinity chromatography, etc.; a method utilizingdifference in hydrophobicity such as reverse phase high performanceliquid chromatography, etc.; a method utilizing difference inisoelectric point such as isoelectrofocusing electrophoresis; and thelike.

When the protein thus obtained is in a free form, the protein can beconverted into the salt by publicly known methods or modificationsthereof. On the other hand, when the protein is obtained in the form ofa salt, it can be converted into the free form or in the form of adifferent salt by publicly known methods or modifications thereof.

The protein produced by the recombinant can be treated, prior to orafter the purification, with an appropriate protein-modifying enzyme sothat the protein can be appropriately modified to partially remove thepolypeptide. Examples of the protein-modifying enzyme include trypsin,chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and thelike.

The presence of the thus produced protein of the present invention canbe determined by an enzyme immunoassay or western blotting using aspecific antibody.

The antibodies to the protein or partial peptide used in the presentinvention, or its salts may be any of polyclonal and monoclonalantibodies, as long as they are capable of recognizing the protein orpartial peptide used in the present invention, or its salts.

The antibodies to the protein or partial peptide used in the presentinvention, or its salts, (hereinafter they are sometimes collectivelyreferred to as the protein of the present invention in the descriptionof the antibodies) can be produced by a publicly known method ofproducing an antibody or antiserum, using the protein of the presentinvention as an antigen.

[Preparation of Monoclonal Antibody]

(a) Preparation of Monoclonal Antibody-Producing Cells

The protein of the present invention is administered to warm-bloodedanimals either solely or together with carriers or diluents to the sitewhere the production of antibody is possible by the administration. Inorder to potentiate the antibody productivity upon the administration,complete Freund's adjuvants or incomplete Freund's adjuvants may beadministered. The administration is usually carried out once every about2 to about 6 weeks and about 2 to about 10 times in total. Examples ofthe applicable warm-blooded animals are monkeys, rabbits, dogs, guineapigs, mice, rats, sheep, goats and fowl, with the use of mice and ratsbeing preferred.

In the preparation of monoclonal antibody-producing cells, warm-bloodedanimals, e.g., mice, immunized with an antigen wherein the antibodytiter is noted are selected, then spleen or lymph node is collectedafter 2 to 5 days from the final immunization and antibody-producingcells contained therein are fused with myeloma cells from homozoic orheterozoic animal to give monoclonal antibody-producing hybridomas.Measurement of the antibody titer in antisera may be carried out, forexample, by reacting a labeled protein, which will be described later,with the antiserum followed by assaying the binding activity of thelabeling agent bound to the antibody. The fusion may be carried out, forexample, by the known method by Koehler and Milstein [Nature, 256, 495,(1975)]. Examples of the fusion accelerator are polyethylene glycol(PEG), Sendai virus, etc., among which PEG is preferably employed.

Examples of the myeloma cells are those collected from warm-bloodedanimals such as NS-1, P3U1, SP2/0, AP-1, etc. In particular, P3U1 ispreferably employed. A preferred ratio of the count of theantibody-producing cells used (spleen cells) to the count of myelomacells is within a range of approximately 1:1 to 20:1. When PEG(preferably, PEG 1000 to PEG 6000) is added in a concentration ofapproximately 10 to 80% followed by incubation at 20 to 40° C.,preferably at 30 to 37° C. for 1 to 10 minutes, an efficient cell fusioncan be carried out.

Various methods can be used for screening of monoclonalantibody-producing hybridomas. Examples of such methods include a methodwhich comprises adding the supernatant of a hybridoma to a solid phase(e.g., a microplate) adsorbed with the protein as an antigen directly ortogether with a carrier, adding an anti-immunoglobulin antibody (wheremouse cells are used for the cell fusion, anti-mouse immunoglobulinantibody is used) labeled with a radioactive substance or an enzyme orProtein A and detecting the monoclonal antibody bound to the solidphase, and a method which comprises adding the supernatant of hybridomato a solid phase adsorbed with an anti-immunoglobulin antibody orProtein A, adding the protein labeled with a radioactive substance or anenzyme and detecting the monoclonal antibody bound to the solid phase,or the like.

The monoclonal antibody can be screened according to publicly knownmethods or their modifications. In general, the screening can beperformed in a medium for animal cells supplemented with HAT(hypoxanthine, aminopterin and thymidine). Any screening and growthmedium can be employed as far as the hybridoma can grow there. Forexample, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20%fetal bovine serum, GIT medium (Wako Pure Chemical Industries, Ltd.)containing 1 to 10% fetal bovine serum, a serum free medium forcultivation of a hybridoma (SFM-101, Nissui Seiyaku Co., Ltd.) and thelike, can be used for the screening and growth medium. The culture iscarried out generally at 20 to 40° C., preferably at 37° C., for about 5days to about 3 weeks, preferably 1 to 2 weeks, normally in 5% CO₂. Theantibody titer of the culture supernatant of a hybridoma can bedetermined as in the assay for the antibody titer in antisera describedabove.

(b) Purification of Monoclonal Antibody

Separation and purification of a monoclonal antibody can be carried outby publicly known methods, such as separation and purification ofimmunoglobulins [for example, salting-out, alcohol precipitation,isoelectric point precipitation, electrophoresis, adsorption anddesorption with ion exchangers (e.g., DEAE), ultracentrifugation, gelfiltration, or a specific purification method which comprises collectingonly an antibody with an activated adsorbent such as an antigen-bindingsolid phase, Protein A or Protein G and dissociating the binding toobtain the antibody.]

[Preparation of Polyclonal Antibody]

The polyclonal antibody of the present invention can be manufactured bypublicly known methods or modifications thereof. For example, awarm-blooded animal is immunized with an immunogen (protein antigen) perse, or a complex of immunogen and a carrier protein is formed and awarm-blooded animal is immunized with the complex in a manner similar tothe method described above for the manufacture of monoclonal antibodies.The product containing the antibody to the protein of the presentinvention is collected from the immunized animal followed by separationand purification of the antibody.

In the complex of immunogen and carrier protein used to immunize awarm-blooded animal, the type of carrier protein and the mixing ratio ofcarrier to hapten may be any type and in any ratio, as long as theantibody is efficiently produced to the hapten immunized by crosslinkingto the carrier. For example, bovine serum albumin, bovine thyroglobulinor hemocyanin is coupled to hapten in a carrier-to-hapten weight ratioof approximately 0.1 to 20, preferably approximately 1 to 5.

A variety of condensation agents can be used for the coupling of carrierto hapten. Glutaraldehyde, carbodiimide, maleimide activated ester andactivated ester reagents containing thiol group or dithiopyridyl groupare used for the coupling.

The condensation product is administered to a warm-blooded animal eithersolely or together with carriers or diluents to the site that canproduce the antibody by the administration. In order to potentiate theantibody productivity upon the administration, complete Freund'sadjuvant or incomplete Freund's adjuvant may be administered. Theadministration is usually made once every about 2 to 6 weeks and about 3to 10 times in total.

The polyclonal antibody can be collected from the blood, ascites, etc.,preferably from the blood of warm-blooded animal immunized by the methoddescribed above.

The polyclonal antibody titer in antiserum can be assayed by the sameprocedure as that for the determination of serum antibody titerdescribed above. The separation and purification of the polyclonalantibody can be carried out, following the method for the separation andpurification of immunoglobulins performed as in the separation andpurification of monoclonal antibodies described hereinabove.

The antisense polynucleotide having a complementary or substantiallycomplementary base sequence to the DNA encoding the protein or partialpeptide used in the present invention (hereinafter these DNAs aresometimes collectively referred to as the DNA of the present inventionin the description of antisense polynucleotide) can be any antisensepolynucleotide, so long as it possesses a base sequence complementary orsubstantially complementary base sequence to that of the DNA of thepresent invention and capable of suppressing expression of the DNA, butantisense DNA is preferred.

The base sequence substantially complementary to the DNA of the presentinvention may include, for example, a base sequence having at leastabout 70% homology, preferably at least about 80% homology, morepreferably at least about 90% homology and most preferably at leastabout 95% homology, to the entire base sequence or the partial basesequence of the base sequence complementary to the DNA of the presentinvention (i.e., complementary strand to the DNA of the presentinvention), and the like. Especially in the entire base sequence of thecomplementary strand to the DNA of the present invention, (a) in thecase of antisense polynucleotide directed to translation inhibition, anantisense polynucleotide having at least about 70% homology, preferablyat least about 80% homology, more preferably at least about 90% homologyand most preferably at least about 95% homology, to the complementarystrand of the base sequence, which encodes the N-terminal region of theprotein of the present invention (e.g., the base sequence around theinitiation codon, etc.), (b) in the case of antisense polynucleotidedirected to RNA cleavage by RNaseH, an antisense polynucleotide havingat least about 70% homology, preferably at least about 80% homology,more preferably at least about 90% homology and most preferably at leastabout 95% homology, to the complementary strand of the entire basesequence of the DNA of the present invention containing introns, aresuitable, respectively.

Specifically, there are an antisense polynucleotide containing theentire or part of a complementary or substantially complementary basesequence to a base sequence of DNA containing the base sequencerepresented by SEQ ID NO: 3 or SEQ ID NO: 4, preferably an antisensepolynucleotide containing the entire or part of a complementary basesequence to a base sequence of DNA containing the base sequencerepresented by SEQ ID NO: 3 or SEQ ID NO: 4 (more preferably, anantisense polynucleotide containing the entire or part of acomplementary base sequence to a base sequence of DNA containing thebase sequence represented by SEQ ID NO: 3, or an antisensepolynucleotide containing the entire or part of a complementary basesequence to a base sequence of DNA containing the base sequencerepresented by SEQ ID NO: 4).

The antisense polynucleotide is generally constituted by bases of about10 to about 40, preferably about 15 to about 30.

To prevent digestion with a hydrolase such as nuclease, etc., thephosphoric acid residue (phosphate) of each nucleotide that constitutesthe antisense DNA may be substituted with chemically modified phosphoricacid residues, e.g., phosphorothioate, methyl phosphonate,phosphorodithionate, etc. Also, the sugar (deoxyribose) in eachnucleotide may be replaced by a chemically modified structure such as2′-O-methylation, etc. The base part (pyrimidine, purine) may also bechemically modified and may be any one which hybridizes to a DNAcontaining the base sequence represented by SEQ ID NO: 2. Theseantisense nucleotides may be synthesized using a publicly known DNAsynthesizer, etc. According to the present invention, the antisensepolynucleotide (nucleic acid) capable of inhibiting the replication orexpression of a gene for the protein of the present invention can bedesigned and synthesized based on the base sequence information ofcloned or identified protein-encoding DNA. Such a polynucleotide(nucleic acid) is hybridizable to RNA of the protein gene of the presentinvention to inhibit the synthesis or function of said RNA or is capableof modulating and/or controlling the expression of the protein gene ofthe present invention via interaction with RNA associated with theprotein of the present invention. Polynucleotides complementary to theselected sequences of RNA associated with the protein of the presentinvention and polynucleotides specifically hybridizable to RNAassociated with the protein of the present invention are useful inmodulating and/or controlling the in vivo and in vitro expression of theprotein gene of the present invention, and are useful for the treatmentor diagnosis of diseases, etc. The term “corresponding” is used to meanhomologous to or complementary to a particular sequence of thenucleotide including the gene, base sequence or nucleic acid. The term“corresponding” between nucleotides, base sequences or nucleic acids andpeptides (proteins) usually refer to amino acids of a peptide (protein)under the order derived from the sequence of nucleotides (nucleic acids)or their complements. In the protein genes, the 5′ end hairpin loop, 5′end 6-base-pair repeats, 5′ end untranslated region, polypeptidetranslation initiation codon, protein coding region, ORF translationtermination codon, 3′ end untranslated region, 3′ end palindrome region,and 3′ end hairpin loop, may be selected as preferred target regions,though any other region may be selected as a target in the proteingenes.

The relationship between the targeted nucleic acids and thepolynucleotides complementary to at least a part of the target region,or the relationship between the target and the hybridizablepolynucleotide can be denoted to be “antisense.” Examples of theantisense polynucleotides include pqlynucleotides containing2-deoxy-D-ribose, polynucleotides containing D-ribose, any other type ofpolynucleotides which are N-glycosides of a purine or pyrimidine base,or other polymers containing non-nucleotide backbones (e.g.,commercially available protein nucleic acids and syntheticsequence-specific nucleic acid polymers) or other polymers containingnonstandard linkages (provided that the polymers contain nucleotideshaving such a configuration that allows base pairing or base stacking,as is found in DNA or RNA), etc. The antisense polynucleotides may bedouble-stranded DNA, single-stranded DNA, double-stranded RNA,single-stranded RNA or a DNA:RNA hybrid, and may further includeunmodified polynucleotides (or unmodified oligonucleotides), those withpublicly known types of modifications, for example, those with labelsknown in the art, those with caps, methylated polynucleotides, thosewith substitution of one or more naturally occurring nucleotides bytheir analogue, those with intramolecular modifications of nucleotidessuch as those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.) and those withcharged linkages or sulfur-containing linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those having side chain groups such asproteins (nucleases, nuclease inhibitors, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), saccharides (e.g., monosaccharides,etc.), those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylating agents, those with modifiedlinkages (e.g., a anomeric nucleic acids, etc.), and the like. Hereinthe terms “nucleoside”, “nucleotide” and “nucleic acid” are used torefer to moieties that contain not only the purine and pyrimidine bases,but also other heterocyclic bases, which have been modified. Suchmodifications may include methylated purines and pyrimidines, acylatedpurines and pyrimidines and other heterocyclic rings. Modifiednucleotides and modified nucleotides also include modifications on thesugar moiety, wherein, for example, one or more hydroxyl groups mayoptionally be substituted with a halogen atom(s), an aliphatic group(s),etc., or may be converted into the corresponding functional groups suchas ethers, amines, or the like.

The antisense polynucleotide (nucleic acid) of the present invention isRNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of themodified nucleic acid are, but not limited to, sulfur and thiophosphatederivatives of nucleic acids and those resistant to degradation ofpolynucleoside amides or oligonucleoside amides. The antisense nucleicacids of the present invention can be modified preferably based on thefollowing design, that is, by increasing the intracellular stability ofthe antisense nucleic acid, increasing the cell permeability of theantisense nucleic acid, increasing the affinity of the nucleic acid tothe targeted sense strand to a higher level, or minimizing the toxicity,if any, of the antisense nucleic acid.

Many of such modifications are known in the art, as disclosed in J.Kawakami, et al., Pharm. Tech. Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp.395, 1992; S. T. Crooke, et al. ed., Antisense Research andApplications, CRC Press, 1993; etc.

The antisense nucleic acid of the present invention may contain alteredor modified sugars, bases or linkages. The antisense nucleic acid mayalso be provided in a specialized form such as liposomes, microspheres,or may be applied to gene therapy, or may be provided in combinationwith attached moieties. Such attached moieties include polycations suchas polylysine that act as charge neutralizers of the phosphate backbone,or hydrophobic moieties such as lipids (e.g., phospholipids,cholesterols, etc.) that enhance the interaction with cell membranes orincrease uptake of the nucleic acid. Preferred examples of the lipids tobe attached are cholesterols or derivatives thereof (e.g., cholesterylchloroformate, cholic acid, etc.). These moieties may be attached to thenucleic acid at the 3′ or 5′ ends thereof and may also be attachedthereto through a base, sugar, or intramolecular nucleoside linkage.Other moieties may be capping groups specifically placed at the 3′ or 5′ends of the nucleic acid to prevent degradation by nucleases such asexonuclease, RNase, etc. Such capping groups include, but are notlimited to, hydroxylprotecting groups known in the art, includingglycols such as polyethylene glycol, tetraethylene glycol and the like.

The inhibitory action of the antisense nucleic acid can be examinedusing the transformant of the present invention, the gene expressionsystem of the present invention in vivo and in vitro, or the translationsystem of the protein in vivo and in vitro. The nucleic acid can beapplied to cells by a variety of publicly known methods.

Hereinafter, the protein of the present invention, its partial peptides,or salts thereof (hereinafter sometimes merely referred to as theprotein of the present invention), the DNA encoding the protein of thepresent invention or its partial peptides (hereinafter sometimes merelyreferred to as the DNA of the present invention), the antibodies to theprotein of the present invention, its partial peptides, or salts thereof(hereinafter sometimes referred to as the antibodies of the presentinvention) and the antisense polynucleotides to the DNA of the presentinvention (hereinafter sometimes merely referred to as the antisensepolynucleotides of the present invention) are specifically described fortheir applications.

The protein of the present invention can be utilized as a disease markersince expression of the protein increases in adipose tissues. That is,the protein is useful as a marker for early diagnosis in insulinsensitization, judgment of severity in conditions, or predicteddevelopment of disease. Furthermore, the protein of the presentinvention has an effect of inducing or aggravating insulin sensitizationto increase blood glucose and blood fat levels. Therefore,pharmaceuticals which comprises the antisense polynucleotide of thepresent invention, the compound or its salt that inhibits the expressionof the protein gene of the present invention or the antibody of thepresent invention can be used, for example, as insulin-sensitizingagents, or further as agents for the prevention and/or treatment ofdiabetes, obesity, hyperlipemia, arteriosclerosis, hypertension or heartdisease.

(1) Screening of Drug Candidate Compounds for Disease

Since expression of the protein of the present invention increases inadipose tissues, the protein has an effect of inducing or aggravatinginsulin sensitization and increases blood glucose and blood fat. Thus,the compound or its salt that inhibits the activity of the protein ofthe present invention can be used, for example, as insulin-sensitizingagents, or as pharmaceuticals for the prevention and/or treatment ofdiabetes, obesity, hyperlipemia, arteriosclerosis, hypertension or heartdisease.

Therefore, the protein of the present invention is useful as a reagentfor screening the compound or its salt that inhibits the activity of theprotein of the present invention.

That is, the present invention provides a method of screening thecompound or its salt that inhibits the activity (e.g., a ligand bindingactivity, a signal transduction activity, etc.) of the protein of thepresent invention, which comprises using the protein of the presentinvention.

More specifically, there is employed a method of screening the compoundor its salt that inhibits the activity of the protein of the presentinvention, which comprises comparing, for example, (i) the ligandbinding activity or signal transduction activity of a cell capable ofproducing the protein of the present invention and (ii) the ligandbinding activity or signal transduction activity of a cell capable ofproducing the protein of the present invention in the presence of a testcompound.

In the screening method described above, for example, in the cases of(i) and (ii), the ligand binding activity or signal transductionactivity can be assayed by publicly known techniques, e.g., by thetechnique described in J. Exp. Med., 194, 1111-1122, 2001, or withmodifications of the technique, and comparison is made.

Specifically, (1) the level of phosphorylated ERK produced, (2) thelevel of TNF-α produced and secreted extracellularly or (3) the level ofphosphorylated protein of the present invention (preferablyphosphorylated TREM-2) produced is determined, respectively, (i) in thecase that a cell (e.g., an animal cell) overexpressed with the proteinof the present invention wherein a vector expressing the protein of thepresent invention has been introduced is cultured, if required by addingto the cell (a) a ligand-containing fluid such as a microorganismlysate, a microorganism supernatant, an eukaryotic cell lysate, aneukaryotic cell supernatant, etc., (b) a ligand itself, (c) a substancehaving a binding activity to the protein of the present inventionequivalent to a naturally occurring ligand or (d) an antibody activatingthe protein of the present invention (e.g., TREM-2) and (ii) in the casethat a cell (e.g., an animal cell) overexpressed with the protein of thepresent invention wherein a vector expressing the protein of the presentinvention has been introduced is cultured in the presence of a testcompound, if required by adding to the cell (a) a ligand-containingfluid such as a microorganism lysate, a microorganism supernatant, aneukaryotic cell lysate, an eukaryotic cell supernatant, etc., (b) aligand itself, (c) a substance having a binding activity to the proteinof the present invention equivalent to a naturally occurring ligand or(d) an antibody activating the protein of the present invention (e.g.,TREM-2), followed by comparison.

The aforesaid level of phosphorylated ERK or the level of TNF-α producedcan be assayed by publicly known methods (e.g., western blotting, EIA,etc.) using an anti-phosphorylated ERK antibody or an anti-TNF-αantibody. The level of phosphorylated TREM-2 produced can be assayed bypublicly known methods (e.g., immunoprecipitation, western blotting,etc.) using an anti-TREM-2 antibody and an anti-phosphorylated tyrosineantibody.

As the cells capable of producing the protein of the present invention,there are used, for example, hosts transformed by a vector containing aDNA encoding the protein of the present invention described above(transformants). As the hosts, animal cells such as COS-7 cells, CHOcells, HEK293 cells, 3T3-L1 cells, L6 cells, C2C12 cells, etc. arepreferably used. For the screening, transformants with the protein ofthe present invention expressed on the cell membrane through incubationby the techniques described above are preferably employed. Thetechniques for incubation of the cell capable of expressing the proteinof the present invention are the same as the techniques for incubationof the transformants of the present invention described above.

Examples of the test compound include peptides, proteins, non-peptidecompounds, synthetic compounds, fermentation products, cell extracts,plant extracts, animal tissue extracts, etc.

For example, when a test compound reduces the ligand binding activity orthe signal transduction activity in the case (i) described above atleast by about 20%, preferably at least by about 30%, more preferably atleast by about 50%, as compared to the case (ii) above, the testcompound can be selected to be a compound capable of inhibiting theactivity of the protein of the present invention.

The compound having the activity of inhibiting the activity of theprotein of the present invention is useful as a safe and low toxicpharmaceutical for suppressing the physiological activity of the proteinof the present invention.

The compound or its salt obtained using the screening method orscreening kit of the present invention is a compound selected from, forexample, peptides, proteins, non-peptide compounds, synthetic compounds,fermentation products, cell extracts, plant extracts, animal tissueextracts, plasma, etc. The salts of the compound used are those givenabove as the salts of the peptide of the present invention.

Furthermore, expression of the gene encoding the protein of the presentinvention also increases in adipose tissues and thus, the compound orits salt that inhibits expression of the gene encoding the protein ofthe present invention can be used as, e.g., an insulin-sensitizingagent, or an agent for the prevention and/or treatment of diabetes,obesity, hyperlipemia, arteriosclerosis, hypertension or heart disease,and so on.

Accordingly, the DNA of the present invention is useful as a reagent forscreening the compound or its salt that inhibits expression of the geneencoding the protein of the present invention.

For the screening method, there is a method of screening, whichcomprises comparing (iii) the case in which the cell capable ofproducing the protein of the present invention is incubated and (iv) thecase in which the cell capable of producing the protein used in thepresent invention is incubated in the presence of a test compound.

In the method described above, the level of the aforesaid gene expressed(specifically, the level of the protein of the present invention or thelevel of mRNA encoding the protein) is measured in the cases (iii) and(iv), and comparison is made therebetween.

As the test compound and the cell capable of producing the protein ofthe present invention, the same examples as described above are given.

The level of the protein can be determined by publicly known methods,e.g., by measuring the aforesaid protein present in the cell extract,etc., using an antibody capable of recognizing the protein of thepresent invention, in accordance with techniques such as westernblotting, EIA, etc., or their modifications.

The level of mRNA can be determined by publicly known techniques, e.g.,Northern hybridization, RT-PCR, or modifications thereof. Alternatively,a reporter gene (e.g., β-galactosidase, etc.) is ligated to a promoterof the gene for the protein of the present invention, the resultingvector is introduced into a cell (e.g., an animal cell, etc.), and thelevel of the reporter gene expressed may be determined.

For example, when a test compound inhibits the level of the geneexpressed in the case (iv) described above at least by about 20%,preferably at least by about 30%, more preferably at least by about 50%,as compared to the case (iii) above, the test compound can be selectedto be a compound capable of inhibiting the expression of the geneencoding the protein of the present invention.

The screening kit of the present invention comprises the protein used inthe present invention, its partial peptide, or a salt thereof, or thecell capable of producing the protein used in the present invention, orits partial peptide.

The compound or its salt obtained using the screening method orscreening kit of the present invention is a compound or its saltselected from the test compounds described above, for example, peptides,proteins, non-peptide compounds, synthetic compounds, fermentationproducts, cell extracts, plant extracts, animal tissue extracts, plasma,etc., and is a compound or its salt, which regulates the activity (e.g.,the ligand binding activity, the signal transduction activity, etc.) ofthe protein of the present invention.

Examples of the salt of the compound are the same as those salts givenfor the protein of the present invention described above.

The compound or its salt that inhibits the activity of the protein ofthe present invention and the compound or its salt that inhibits theexpression of the gene encoding the protein of the present invention arerespectively useful as pharmaceuticals such as insulin-sensitizingagents, agents for the prevention and/or treatment of diabetes, obesity,hyperlipemia, arteriosclerosis, hypertension or heart disease, and soon.

Where the compound or its salt obtained using the screening method orscreening kit of the present invention is used as the agent for theprevention and/or treatment described above, the compound or its saltcan be prepared into a pharmaceutical composition in a conventionalmanner.

For example, the composition for oral administration includes solid orliquid preparations, specifically, tablets (including dragees andfilm-coated tablets), pills, granules, powdery preparations, capsules(including soft capsules), syrup, emulsions, suspensions, etc. Such acomposition is manufactured by publicly known methods and contains avehicle, a diluent or an excipient conventionally used in the field ofpharmaceutical preparations. Examples of the vehicle or excipient fortablets are lactose, starch, sucrose, magnesium stearate, etc.

Examples of the composition for parenteral administration are injectablepreparations, suppositories, etc. The injectable preparations mayinclude dosage forms such as intravenous injection, subcutaneousinjection, intracutaneous injection, intramuscular injection, dripinfusion, intraarticular injection, etc. These injectable preparationsmay be prepared by methods publicly known in the art. For example, theinjectable preparations may be prepared by dissolving, suspending oremulsifying the compound or its salt described above in a sterileaqueous medium or an oily medium conventionally used for injections. Asthe aqueous medium for injections, there are, for example, physiologicalsaline, an isotonic solution containing glucose and other auxiliaryagents, etc., which may be used in combination with an appropriatedissolution aid such as an alcohol (e.g., ethanol), a polyalcohol (e.g.,propylene glycol, polyethylene glycol), a nonionic surfactant [e.g.,polysorbate 80, HCO-50 (polyoxyethylene (50 mols) adduct of hydrogenatedcastor oil)], etc. As the oily medium, there are employed, e.g., sesameoil, soybean oil, etc., which may be used in combination with adissolution aid such as benzyl benzoate, benzyl alcohol, etc. Theinjectable preparation thus prepared is preferably filled in anappropriate ampoule. The suppository used for rectal administration maybe prepared by blending the aforesaid compound or its salt withconventional bases for suppositories.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into pharmaceutical preparations with aunit dose suited to fit a dose of the active ingredients. Such unit dosepreparations include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid compoundcontained is generally 1 to 500 mg per each dosage unit form; especiallyin the form of injection, it is preferred that the aforesaid compound iscontained in approximately 0.5 to 100 mg.

Each composition described above may further contain other activecomponents, unless formulation with the compound described above causesany adverse interaction.

Since the pharmaceutical preparations thus obtained are safe and lowtoxic, they can be administered to, e.g., human or a warm-blooded animal(e.g., mouse, rat, rabbit, sheep, swine, bovine, horse, fowl, cat, dog,monkey, chimpanzee, etc.) orally or parenterally.

The dose of the compound or its salt may vary depending upon its action,target disease, subject to be administered, route of administration,etc. For example, when the compound or its salt that inhibits theactivity of the protein of the present invention is orally administeredfor the purpose of sensitizing insulin, the compound or its salt isgenerally administered to an adult (as 60 kg body weight) in a dailydose of about 0.1 to about 100 mg, preferably about 1.0 to about 50 mgand more preferably about 1.0 to about 20 mg. In parenteraladministration, a single dose of the said compound or its salt may varydepending upon subject to be administered, target disease, etc. When thecompound or its salt that inhibits the activity of the protein of thepresent invention is administered to an adult (as 60 kg body weight) inthe form of an injectable preparation for the purpose of sensitizinginsulin, it is advantageous to administer the compound or its saltintravenously in a daily dose of about 0.01 to about 30 mg, preferablyabout 0.1 to about 20 mg, and more preferably about 0.1 to about 10 mg.For other animal species, the corresponding dose as converted per 60 kgweight can be administered.

(2) Quantification for the Protein of the Present Invention, it PartialPeptide or Salts Thereof.

The antibody to the protein of the present invention (hereinaftersometimes merely referred to as the antibody of the present invention)is capable of specifically recognizing the protein of the presentinvention, and thus can be used for quantification of the protein of thepresent invention in a test sample fluid, in particular, forquantification by sandwich immunoassay; etc.

That is, the present invention provides:

-   (i) a method for quantification of the protein of the present    invention in a test sample fluid, which comprises competitively    reacting the antibody of the present invention, a test sample fluid    and the labeled form of the protein of the present invention, and    measuring the ratio of the labeled form of the protein of the    present invention bound to said antibody; and,-   (ii) a method for quantification of the protein of the present    invention in a test sample fluid, which comprises reacting a test    sample fluid simultaneously or continuously with the antibody of the    present invention immobilized on a carrier and another labeled    antibody of the present invention, and then measuring the activity    of the labeling agent on the insoluble carrier.

In the quantification method (ii) described above, it is preferred thatone antibody is capable of recognizing the N-terminal region of theprotein of the present invention, while another antibody is capable ofreacting with the C-terminal region of the protein of the presentinvention.

The monoclonal antibody to the protein of the present invention(hereinafter sometimes referred to as the monoclonal antibody of thepresent invention) can be used to quantify the protein of the presentinvention. In addition, the protein can be detected by means of a tissuestaining as well. For these purposes, the antibody molecule per se maybe used or F(ab′)₂, Fab′ or Fab fractions of the antibody molecule mayalso be used.

The method for quantification of the protein of the present inventionusing the antibody of the present invention is not particularly limited.Any quantification method can be used, so long as the amount ofantibody, antigen or antibody-antigen complex corresponding to theamount of antigen (e.g., the amount of the protein) in a test samplefluid can be detected by chemical or physical means and the amount ofthe antigen can be calculated from a standard curve prepared fromstandard solutions containing known amounts of the antigen. For such anassay method, for example, nephrometry, the competitive method, theimmunometric method, the sandwich method, etc. are suitably used and interms of sensitivity and specificity, it is particularly preferred touse the sandwich method described hereinafter.

Examples of the labeling agent used in the assay method using thelabeling substance are radioisotopes, enzymes, fluorescent substances,luminescent substances, and the like. As the radioisotopes, there areused, e.g., [¹²⁵I], [¹³¹I], [³H], [¹⁴C], etc. The enzymes describedabove are preferably enzymes, which are stable and have a high specificactivity, and include, e.g., β-galactosidase, β-glucosidase, an alkalinephosphatase, a peroxidase, malate dehydrogenase, etc. As the fluorescentsubstances, there are used, e.g., fluorescamine, fluoresceinisothiocyanate, etc. As the luminescent substances described above thereare used, e.g., luminol, a luminol derivative, luciferin, lucigenin,etc. Furthermore, the biotin-avidin system may be used as well forbinding of an antibody or antigen to a labeling agent.

For immobilization of the antigen or antibody, physical adsorption maybe used. Chemical binding techniques conventionally used forinsolubilization or immobilization of proteins, enzymes, etc. may alsobe used. For carriers, there are used, e.g., insoluble polysaccharidessuch as agarose, dextran, cellulose, etc.; synthetic resin such aspolystyrene, polyacrylamide, silicon, etc., and glass or the like.

In the sandwich method, the immobilized monoclonal antibody of thepresent invention is reacted with a test sample fluid (primaryreaction), then with a labeled form of another monoclonal antibody ofthe present invention (secondary reaction), and the activity of thelabel on the immobilizing carrier is measured, whereby the amount of theprotein of the present invention in the test sample fluid can bequantified. The order of the primary and secondary reactions may bereversed, and the reactions may be performed simultaneously or with aninterval. The methods of labeling and immobilization can be performed bythe methods described above. In the immunoassay by the sandwich method,the antibody used for immobilized or labeled antibodies is notnecessarily one species, but a mixture of two or more species ofantibody may be used to increase the measurement sensitivity.

In the methods of determining the protein of the present invention bythe sandwich method, antibodies that bind to different sites of theprotein of the present invention are preferably used as the monoclonalantibodies of the present invention used for the primary and secondaryreactions. That is, in the antibodies used for the primary and secondaryreactions are, for example, when the antibody used in the secondaryreaction recognizes the C-terminal region of the protein of the presentinvention, it is preferable to use the antibody recognizing the regionother than the C-terminal region for the primary reaction, e.g., theantibody recognizing the N-terminal region.

The monoclonal antibodies of the present invention can be used for theassay systems other than the sandwich method, for example, thecompetitive method, the immunometric method, nephrometry, etc.

In the competitive method, antigen in a test sample fluid and thelabeled antigen are competitively reacted with antibody, and theunreacted labeled antigen (F) and the labeled antigen bound to theantibody (B) are separated (B/F separation). The amount of the label inB or F is measured, and the amount of the antigen in the test samplefluid is quantified. This reaction method includes a liquid phase methodusing a soluble antibody as an antibody, polyethylene glycol for B/Fseparation and a secondary antibody to the soluble antibody, and animmobilized method either using an immobilized antibody as the primaryantibody, or using a soluble antibody as the primary antibody andimmobilized antibody as the secondary antibody.

In the immunometric method, antigen in a test sample fluid andimmobilized antigen are competitively reacted with a definite amount oflabeled antibody, the immobilized phase is separated from the liquidphase, or antigen in a test sample fluid and an excess amount of labeledantibody are reacted, immobilized antigen is then added to bind theunreacted labeled antibody to the immobilized phase, and the immobilizedphase is separated from the liquid phase. Then, the amount of the labelin either phase is measured to quantify the antigen in the test samplefluid.

In the nephrometry, insoluble precipitate produced after theantigen-antibody reaction in gel or solution is quantified. When theamount of antigen in the test fluid is small and only a small amount ofprecipitate is obtained, laser nephrometry using scattering of laser isadvantageously employed.

For applying these immunological methods to the measurement methods ofthe present invention, any particular conditions or procedures are notrequired. Systems for measuring the protein of the present invention orits salts are constructed by adding the usual technical consideration inthe art to the conventional conditions and procedures. For the detailsof these general technical means, reference can be made to the followingreviews and texts.

For example, Hiroshi Irie, ed. “Radioimmunoassay” (Kodansha, publishedin 1974), Hiroshi Irie, ed. “Sequel to the Radioimmunoassay” (Kodansha,published in 1979), Eiji Ishikawa, et al. ed. “Enzyme immunoassay”(Igakushoin, published in 1978), Eiji Ishikawa, et al. ed. “Immunoenzymeassay” (2nd ed.) (Igakushoin, published in 1982), Eiji Ishikawa, et al.ed. “Immunoenzyme assay” (3rd. ed.) (Igakushoin, published in 1987),Methods in ENZYMOLOGY, Vol. 70 (Immunochemical Techniques (Part A)),ibid., Vol. 73 (Immunochemical Techniques (Part B)), ibid., Vol. 74(Immunochemical Techniques (Part C)), ibid., Vol. 84 (ImmunochemicalTechniques (Part D: Selected Immunoassays)), ibid., Vol. 92(Immunochemical Techniques (Part E: Monoclonal Antibodies and GeneralImmunoassay Methods)), ibid., Vol. 121 (Immunochemical Techniques (PartI: Hybridoma Technology and Monoclonal Antibodies)) (all published byAcademic Press Publishing).

As described above, the protein of the present invention can bequantified with high sensitivity, using the antibody of the presentinvention.

Furthermore, when an increased level or decreased level of the proteinof the present invention is detected by quantifying the level of theprotein of the present invention using the antibody of the presentinvention, it can be diagnosed that one suffers from insulinsensitization, diabetes, obesity, hyperlipemia, arteriosclerosis,hypertension or heart disease, etc., or it is highly likely to sufferfrom these disease in the future.

Moreover, the antibody of the present invention can be used to detectthe protein of the present invention, which is present in a test samplefluid such as a body fluid, a tissue, etc. The antibody can also be usedto prepare an antibody column for purification of the protein of thepresent invention, detect the protein of the present invention in eachfraction upon purification, analyze the behavior of the protein of thepresent invention in the cells under investigation; etc.

(3) Gene Diagnostic Agent

By using the DNA of the present invention, e.g., as a probe, anabnormality (gene abnormality) of the DNA or mRNA encoding the proteinof the present invention in human or warm-blooded animal (e.g., rat,mouse, guinea pig, rabbit, fowl, sheep, swine, bovine, horse, cat, dog,monkey, chimpanzee, etc.) can be detected. Thus, the DNA of the presentinvention is useful as a gene diagnostic agent for detecting damages tothe DNA or mRNA, its mutation, or decreased expression, increasedexpression, overexpression, etc. of the DNA or mRNA, and so on.

The gene diagnosis described above using the DNA of the presentinvention can be performed by, for example, the Northern hybridizationpublicly known in the art or the PCR-SSCP assay (Genomics, 5, 874-879,1989, Proceedings of the National Academy of Sciences of the UnitedStates of America, 86, 2766-2770, 1989), etc.

For example, when overexpression or decreased expression is detected byNorthern hybridization or DNA mutation is detected by the PCR-SSCPassay, it can be diagnosed that it is highly likely to suffer from,e.g., diabetes, obesity, hyperlipemia, arteriosclerosis, hypertension orheart disease, and so on.

(4) Pharmaceutical which Comprises the Extracellular Domain of theProtein of the Present Invention or Its Salt

The extracellular domain of the protein of the present invention or itssalt has the activity of binding to the ligand of the protein of thepresent invention but has no signal transduction activity, and can thussuppress signal transduction of the protein of the present invention asa dominant negative protein. The extracellular domain of the protein ofthe present invention or its salt has an effect of sensitizing insulinto reduce blood glucose and blood fat.

Therefore, the extracellular domain of the protein of the presentinvention or its salt is useful as, e.g., an insulin-sensitizing agentand further as a pharmaceutical such as an agent for the preventionand/or treatment of diabetes, obesity, hyperlipemia, arteriosclerosis,hypertension or heart disease, and so on.

The extracellular domain of the protein of the present invention may beadministered singly as it is, or as an appropriate pharmaceuticalcomposition. The pharmaceutical composition used for the administrationcomprises the aforesaid extracellular domain or its salt and apharmacologically acceptable carrier, a diluent or excipient. Such acomposition is provided as a pharmaceutical composition suitable fororal or parenteral administration (e.g., subcutaneous administration).

The extracellular domain described above can be used orally, forexample, in the form of tablets which may be sugar coated if necessary,capsules, elixirs, microcapsules, etc., or parenterally in the form ofinjectable preparations such as a sterile solution, a suspension, etc.in water or with other pharmaceutically acceptable liquid. Thesepreparations can be prepared by mixing the polypeptide of the presentinvention with a physiologically acceptable known carrier, a flavoringagent, an excipient, a vehicle, an antiseptic agent, a stabilizer, abinder, etc. in a unit dosage form required in a generally acceptedmanner that is applied to making pharmaceutical preparations. The activeingredient in the preparation is controlled in such a dose that anappropriate dose is obtained within the specified range given.

Additives which can be blended with tablets, capsules, etc. include abinder such as gelatin, corn starch, tragacanth and gum arabic, anexcipient such as crystalline cellulose, a swelling agent such as cornstarch, gelatin and alginic acid, a lubricant such as magnesiumstearate, a sweetening agent such as sucrose, lactose and saccharin, anda flavoring agent such as peppermint, akamono oil or cherry. When theunit dosage is in the form of capsules, liquid carriers such as oils andfats may further be used together with the additives described above. Asterile composition for injection may be formulated by conventionalprocedures used to make pharmaceutical compositions, e.g., by dissolvingor suspending the active ingredients in a vehicle such as water forinjection with a naturally occurring vegetable oil such as sesame oiland coconut oil, etc. to prepare the pharmaceutical composition.

Examples of an aqueous medium for injection include physiological salineand an isotonic solution containing glucose and other auxiliary agents(e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) and may be used incombination with an appropriate dissolution aid such as an alcohol(e.g., ethanol or the like), a polyalcohol (e.g., propylene glycol,polyethylene glycol, etc.), a nonionic surfactant (e.g., polysorbate80™, HCO-50, etc.), etc. Examples of the oily medium include sesame oiland soybean oil, which may also be used in combination with adissolution aid such as benzyl benzoate, benzyl alcohol, etc. Thepharmaceuticals may also be formulated with a buffer (e.g., phosphatebuffer, sodium acetate buffer, etc.), a soothing agent (e.g.,benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer(e.g., human serum albumin, polyethylene glycol, etc.), a preservative(e.g., benzyl alcohol, phenol, etc.), an antioxidant, etc. The thusprepared liquid for injection is normally filled in an appropriateampoule.

As the pharmaceutical preparation described above is low toxic, it canbe administered to human or a mammal (e.g., rat, rabbit, sheep, swine,bovine, cat, dog, monkey, etc.) orally or parenterally (e.g.,subcutaneously), directly as a liquid preparation or as a pharmaceuticalcomposition in an appropriate dosage form. The dose may vary dependingupon subject to be administered, target disease, condition, route ofadministration, etc. For example, when the pharmaceutical is used forthe purpose of improving adult insulin sensitization, it is advantageousto administer the domain described above as an injectable preparation ina single dose of about 0.01 to about 20 mg/kg body weight, preferablyabout 0.1 to about 10 mg/kg body weight, more preferably about 0.1 toabout 5 mg/kg body weight, in about 1 to about 5 times per day,preferably in about 1 to about 3 times per day. In other parenteraladministration and oral administration, the domain can be administeredin a dose corresponding to the above dose. When the condition isespecially severe, the dose may be increased according to the condition.

(5) Pharmaceutical which Comprises an Antisense Polynucleotide

The antisense polynucleotide of the present invention that binds to theDNA of the present invention complementarily to inhibit expression ofthe DNA is low toxic and can suppress the function (e.g., the ligandbinding activity or the signal transduction activity) of the protein ofthe present invention or the DNA of the present invention in vivo. Theantisense polynucleotide of the present invention has the action ofsensitizing insulin to reduce blood glucose and blood fat.

Therefore, the antisense polynucleotide of the present invention can beused as an insulin-sensitizing agent and further as an agent for theprevention and/or treatment of obesity, hyperlipemia, arteriosclerosis,hypertension or heart disease, etc.

Where the above antisense polynucleotide is used as the agent for theprevention and/or treatment described above, it can be prepared intopharmaceutical preparations by publicly known techniques, which areprovided for administration.

For example, the above antisense polynucleotide is administereddirectly, or the antisense polynucleotide is inserted into anappropriate vector such as retrovirus vector, adenovirus vector,adenovirus-associated virus vector, etc., followed by treating in aconventional manner. The antisense polynucleotide may then beadministered orally or parenterally to human or mammal (e.g., rat,rabbit, sheep, swine, bovine, cat, dog, monkey, etc.) in a conventionalmanner. The antisense polynucleotide may also be administered directlyas it stands, or may be prepared in pharmaceutical preparations togetherwith a physiologically acceptable carrier to assist its uptake, whichare then administered by gene gun or through a catheter such as acatheter with a hydrogel. Alternatively, the antisense polynucleotidemay be prepared into an aerosol, which is topically administered intothe trachea as an inhaler.

Further for purposes of improving pharmacokinetics, prolonging ahalf-life and improving intracellular uptake efficiency, the antisensepolynucleotide described above is prepared into pharmaceuticalpreparations (injectable preparations) alone or together with a carriersuch as liposome, etc. and the preparations may be administeredintravenously, subcutaneously, intraarticularly, etc.

A dose of the antisense polynucleotide may vary depending on targetdisease, subject to be administered, route for administration, etc. Forexample, where the antisense polynucleotide of the present invention isadministered for the purpose of sensitizing insulin, the antisensepolynucleotide is generally administered to an adult (60 kg body weight)in a daily dose of about 0.1 to 100 mg.

In addition, the antisense polynucleotide may also be used as anoligonucleotide probe for diagnosis to examine the presence of the DNAof the present invention in tissues or cells and states of itsexpression.

As the antisense polynucleotide described above can, the double-strandedRNA containing a part of RNA encoding the protein of the presentinvention, ribozyme containing a part of RNA encoding the protein of thepresent invention, etc. can also prevent expression of the gene of thepresent invention to suppress the in vivo function of the protein usedin the present invention or the DNA used in the present invention andthus can be used as, e.g., an insulin-sensitizing agent, an agent forthe prevention and/or treatment of obesity, hyperlipemia,arteriosclerosis, hypertension or heart disease, etc.

The double-stranded RNA can be designed based on a sequence of thepolynucleotide of the present invention and manufactured bymodifications of publicly known methods (e.g., Nature, 411, 494, 2001).

The ribozyme can be designed based on a sequence of the polynucleotideof the present invention and manufactured by modifications of publiclyknown methods (e.g., TRENDS in Molecular Medicine, 7, 221, 2001). Forexample, the ribozyme can be manufactured by ligating a publicly knownribozyme to a part of the RNA encoding the protein of the presentinvention. A part of the RNA encoding the protein of the presentinvention includes a portion proximal to a cleavage site on the RNA ofthe present invention, which may be cleaved by a publicly known ribozyme(RNA fragment).

Where the double-stranded RNA or ribozyme described above is used as theagent for prevention and/or treatment described above, thedouble-stranded RNA or ribozyme is prepared into pharmaceuticalpreparations as in the antisense polynucleotide, and the preparationscan be provided for administration.

(6) Pharmaceutical which Comprises the Antibody of the Present Invention

The antibody having the action of neutralizing the activity of theprotein of the present invention has the action of sensitizing insulinto reduce blood glucose and blood fat.

Therefore, the antibody (preferably, neutralizing antibody) of thepresent invention can be used as, e.g., an insulin-sensitizing agent, orfurther as a pharmaceutical such as an agent for the prevention and/ortreatment of obesity, hyperlipemia, arteriosclerosis, hypertension orheart disease, etc.

As the aforesaid pharmaceutical containing the antibody of the presentinvention is low toxic, the pharmaceutical can be administered to humanor mammals (e.g., rats, rabbits, sheep, swine, bovine, cats, dogs,monkeys, etc.) orally or parenterally (e.g., intravenously), directly inthe form of a liquid preparation, or as a pharmaceutical composition inan appropriate dosage form. The dose may vary depending upon subject tobe administered, target disease, condition, route of administration,etc. For example, when the pharmaceutical preparation is used for thepurpose of improving adult insulin sensitization, it is advantageous toadminister the antibody of the present invention as an injectablepreparation in a single dose of about 0.01 to about 20 mg/kg bodyweight, preferably about 0.1 to about 10 mg/kg body weight, morepreferably about 0.1 to about 5 mg/kg body weight, in about 1 to about 5times per day, preferably in about 1 to about 3 times per day. In otherparenteral administration and oral administration, the antibody can beadministered in a dose corresponding to the above dose. When thecondition is especially severe, the dose may be increased according tothe condition.

The antibody of the present invention can be administered directly as itis or as an appropriate pharmaceutical composition. The pharmaceuticalcomposition used for the administration described above comprises theantibody or its salt described above and a pharmacologically acceptablecarrier, a diluent or excipient. Such a composition is provided as adosage form suitable for oral or parenteral administration (e.g.,intravenous administration).

Each composition described above may further contain other activecomponents, unless formulation with the antibody described above causesany adverse interaction.

(7) DNA Transgenic Animal

The present invention provides a non-human mammal bearing DNA encodingthe protein of the present invention, which is exogenous (hereinafterabbreviated as the exogenous DNA of the present invention) or itsvariant DNA (sometimes simply referred to as the exogenous variant DNAof the present invention).

That is, the present invention provides:

-   (1) A non-human mammal bearing the exogenous DNA of the present    invention or its variant DNA;-   (2) The mammal according to (1), wherein the non-human mammal is a    rodent;-   (3) The mammal according to (2), wherein the rodent is mouse or rat;    and,-   (4) A recombinant vector containing the exogenous DNA of the present    invention or its variant DNA and capable of expressing in a mammal;    etc.

The non-human mammal bearing the exogenous DNA of the present inventionor its variant DNA (hereinafter simply referred to as the DNA transgenicanimal of the present invention) can be prepared by transfecting adesired DNA into an unfertilized egg, a fertilized egg, a spermatozoon,a germinal cell containing a primordial germinal cell thereof, or thelike, preferably in the embryogenic stage in the development of anon-human mammal (more preferably in the single cell or fertilized cellstage and generally before the 8-cell phase), by standard means, such asthe calcium phosphate method, the electric pulse method, the lipofectionmethod, the agglutination method, the microinjection method, theparticle gun method, the DEAE-dextran method, etc. Also, it is possibleto transfect the exogenous DNA of the present invention into a somaticcell, a living organ, a tissue cell, or the like by the DNA transfectionmethods, and utilize the transformant for cell culture, tissue culture,etc. In addition, these cells may be fused with the above-describedgerminal cell by a publicly known cell fusion method to prepare the DNAtransgenic animal of the present invention.

Examples of the non-human mammal that can be used include bovine, swine,sheep, goat, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats,etc. Above all, preferred are rodents, especially mice (e.g., C57B1/6strain, DBA2 strain, etc. for a pure line and for a cross line, B6C3F₁strain, BDF₁ strain B6D2F₁ strain, BALB/c strain, ICR strain, etc.),rats (Wistar, SD, etc.) or the like, since they are relatively short inontogeny and life cycle from a standpoint of creating model animals forhuman disease.

“Mammals” in a recombinant vector that can be expressed in the mammalsinclude the aforesaid non-human mammals and human.

The exogenous DNA of the present invention refers to the DNA of thepresent invention that is once isolated and extracted from mammals, notthe DNA of the present invention inherently possessed by the non-humanmammals.

The mutant DNA of the present invention includes mutants resulting fromvariation (e.g., mutation, etc.) in the base sequence of the originalDNA of the present invention, specifically DNAs resulting from baseaddition, deletion, substitution with other bases, etc. and furtherincluding abnormal DNA.

The abnormal DNA is intended to mean DNA that expresses the abnormalprotein of the present invention and exemplified by the DNA thatexpresses a protein for suppressing the function of the normal proteinof the present invention.

The exogenous DNA of the present invention may be any one of thosederived from a mammal of the same species as, or a different speciesfrom, the mammal as the target animal. In transfecting the DNA of thepresent invention, it is generally advantageous to use the DNA as a DNAconstruct in which the DNA is ligated downstream a promoter capable ofexpressing the DNA in the target animal. For example, in the case oftransfecting the human DNA of the present invention, a DNA transgenicmammal that expresses the DNA of the present invention to a high level,can be prepared by microinjecting a DNA construct (e.g., vector, etc.)ligated with the human DNA of the present invention into a fertilizedegg of the target non-human mammal downstream various promoters whichare capable of expressing the DNA derived from various mammals (e.g.,rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) bearingthe DNA of the present invention highly homologous to the human DNA.

As expression vectors for the protein of the present invention, thereare Escherichia coli-derived plasmids, Bacillus subtilis-derivedplasmids, yeast-derived plasmids, bacteriophages such as λ phage,retroviruses such as Moloney leukemia virus, etc., and animal virusessuch as vaccinia virus, baculovirus, etc. Of these vectors, Escherichiacoli-derived plasmids, Bacillus subtilis-derived plasmids, oryeast-derived plasmids, etc. are preferably used.

Examples of these promoters for regulating the DNA expression describedabove include (1) promoters for DNA derived from viruses (e.g., simianvirus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancervirus, poliovirus, etc.), and (2) promoters derived from various mammals(human, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.),for example, promoters of albumin, insulin II, uroplakin II, elastase,erythropoietin, endothelin, muscular creatine kinase, glial fibrillaryacidic protein, glutathione S-transferase, platelet-derived growthfactor β, keratins K1, K10 and K14, collagen types I and II, cyclicAMP-dependent protein kinase βI subunit, dystrophin, tartarate-resistantalkaline phosphatase, atrial natriuretic factor, endothelial receptortyrosine kinase (generally abbreviated as Tie2), sodium-potassiumadenosine triphosphorylase (Na,K-ATPase), neurofilament light chain,metallothioneins I and IIA, metalloproteinase I tissue inhibitor, MHCclass I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroidperoxidase (TPO), protein chain elongation factor 1α (EF-1α), β actin, αand β myosin heavy chains, myosin light chains 1 and 2, myelin baseprotein, thyroglobulins, Thy-1, immunoglobulins, H-chain variable region(VNP), serum amyloid component P, myoglobin, troponin C, smooth muscle aactin, preproencephalin A, vasopressin, etc. Among them, cytomegaloviruspromoters, human protein elongation factor 1α (EF-1α) promoters, humanand fowl β actin promoters, etc., which are capable of high expressionin the whole body are preferred.

Preferably, the vectors described above have a sequence that terminatesthe transcription of the desired messenger RNA in the DNA transgenicanimal (generally termed a terminator); for example, a sequence of eachDNA derived from viruses and various mammals, and SV40 terminator of thesimian virus and the like are preferably used.

In addition, for the purpose of increasing the expression of the desiredexogenous DNA to a higher level, the splicing signal and enhancer regionof each DNA, a portion of the intron of an eukaryotic DNA may also beligated at the 5′ upstream of the promoter region, or between thepromoter region and the translational region, or at the 3′ downstream ofthe translational region, depending upon purposes.

The translational region for the normal protein of the present inventioncan be obtained using as a starting material the entire genomic DNA orits portion of liver, kidney, thyroid cell or fibroblast origin fromhuman or various mammals (e.g., rabbits, dogs, cats, guinea pigs,hamsters, rats, mice, etc.) or of various commercially available genomicDNA libraries, or using cDNA prepared by a publicly known method fromRNA of liver, kidney, thyroid cell or fibroblast origin as a startingmaterial. Also, an exogenous abnormal DNA can produce the translationalregion through variation of the translational region of normal proteinobtained from the cells or tissues described above by point mutagenesis.

The translational region can be prepared by a conventional DNAengineering technique, in which the DNA is ligated downstream theaforesaid promoter and if desired, upstream the translation terminationsite, as a DNA construct capable of being expressed in the transgenicanimal.

The exogenous DNA of the present invention is transfected at thefertilized egg cell stage in a manner such that the DNA is certainlypresent in all the germinal cells and somatic cells of the targetmammal. The fact that the exogenous DNA of the present invention ispresent in the germinal cells of the animal prepared by DNA transfectionmeans that all offspring of the produced animal will retain theexogenous DNA of the present invention in all of the germinal cells andsomatic cells thereof. The offspring of the animal that inherits theexogenous DNA of the present invention also have the exogenous DNA ofthe present invention in all of the germinal cells and somatic cellsthereof.

The non-human mammal in which the normal exogenous DNA of the presentinvention has been transfected can be passaged as the DNA-bearing animalunder ordinary rearing environment, after confirming that the exogenousDNA is stably retained by crossing.

By transfection of the exogenous DNA of the present invention at thefertilized egg cell stage, the DNA is retained to be excess in all ofthe germinal and somatic cells. The fact that the exogenous DNA of thepresent invention is excessively present in the germinal cells of theprepared animal after transfection means that the DNA of the presentinvention is excessively present in all of the germinal cells andsomatic cells thereof. The offspring of the animal that inherits theexogenous DNA of the present invention have excessively the DNA of thepresent invention in all of the germinal cells and somatic cellsthereof.

A homozygous animal having the introduced DNA on both of homologouschromosomes can be acquired and male and female of the animal can bebred so that all the progeny retain the DNA in excess.

In a non-human mammal bearing the normal DNA of the present invention,the normal DNA of the present invention has expressed at a high level,and may eventually develop hyperfunction in the function of the proteinof the present invention by accelerating the function of endogenousnormal DNA. Therefore, the animal can be utilized as a pathologic modelanimal for such a disease. For example, using the normal DNA transgenicanimal of the present invention, it is possible to elucidate themechanism of hyperfunction in the function of the protein of the presentinvention and the pathological mechanism of the disease associated withthe protein of the present invention and to investigate how to treatthese diseases.

Furthermore, since a mammal transfected with the exogenous normal DNA ofthe present invention exhibits an increasing symptom of the protein ofthe present invention liberated, the animal is also usable for ascreening test of an agent for the prevention and/or treatment ofdiseases associated with the protein of the present invention, forexample, an insulin-sensitizing agent or an agent for the preventionand/or treatment of obesity, hyperlipemia, arteriosclerosis,hypertension or heart disease.

On the other hand, a non-human mammal having the exogenous abnormal DNAof the present invention can be passaged under normal breedingconditions as the DNA-bearing animal after confirming stable retentionof the exogenous DNA via crossing. Furthermore, the exogenous DNA ofinterest can be utilized as a starting material by inserting the DNAinto the plasmid described above. The DNA construct with a promoter canbe prepared by conventional DNA engineering techniques. The transfectionof the abnormal DNA of the present invention at the fertilized egg cellstage is preserved to be present in all of the germinal and somaticcells of the target mammal. The fact that the abnormal DNA of thepresent invention is present in the germinal cells of the animal afterDNA transfection means that all of the offspring of the prepared animalhave the abnormal DNA of the present invention in all of the germinaland somatic cells. Such an offspring that inherited the exogenous DNA ofthe present invention will have the abnormal DNA of the presentinvention in all of the germinal and somatic cells. A homozygous animalhaving the introduced DNA on both of homologous chromosomes can beacquired, and by crossing these male and female animals, all theoffspring can be bred to retain the DNA.

In a non-human mammal bearing the abnormal DNA of the present invention,the abnormal DNA of the present invention has expressed to a high level,and may eventually develop the function inactive type inadaptability tothe protein of the present invention by inhibiting the functions ofendogenous normal DNA. Therefore, the animal can be utilized as apathologic model animal for such a disease. For example, using thetransgenic animal expressing the abnormal DNA of the present invention,it is possible to elucidate the mechanism of the function inactive typeinadaptability to the protein of the present invention and thepathological mechanism of the disease associated with the protein of thepresent invention and to investigate how to treat the disease.

Specifically, the transgenic animal of the present invention expressingthe abnormal DNA of the present invention at a high level is expected toserve as an experimental model to elucidate the mechanism of thefunctional inhibition (dominant negative effect) of a normal protein bythe abnormal protein of the present invention in the function inactivetype inadaptability of the protein of the present invention.

Moreover, a mammal bearing the abnormal exogenous DNA of the presentinvention has a condition of increasing a free form of the protein ofthe present invention, and is also available for a screening test of theprotein of the present invention or an agent for the improvement or forthe prevention and/or treatment of the function inactive typeinadaptability, for example, an insulin-sensitizing agent, or an agentfor the prevention and/or treatment of obesity, hyperlipemia,arteriosclerosis, hypertension or heart disease.

Other potential applications of two kinds of the DNA transgenic animalsof the present invention described above further include:

-   (1) Use as a cell source for tissue culture;-   (2) Elucidation of the relation to a peptide that is specifically    expressed or activated by the protein of the present invention, by    direct analysis of DNA or RNA in tissues of the DNA transgenic    animal of the present invention or by analysis of the peptide    tissues expressed by the DNA;-   (3) Research on the function of cells derived from tissues that are    usually cultured only with difficulty, using cells in tissues    bearing the DNA cultured by a standard tissue culture technique;-   (4) Screening a drug that enhances the functions of cells using the    cells described in (3) above; and,-   (5) Isolation and purification of the variant protein of the present    invention and preparation of an antibody thereto.

Furthermore, clinical conditions of a disease associated wit the proteinof the present invention, including the function inactive typeinadaptability to the protein of the present invention can be determinedby using the DNA transgenic animal of the present invention. Also,pathological findings on each organ in a disease model associated withthe protein of the present invention can be obtained in more detail,leading to the development of a new method for treatment as well as theresearch and therapy of any secondary diseases associated with thedisease.

Further by withdrawing each organ from the DNA transgenic animal of thepresent invention, mincing the organ and degrading with a proteinasesuch as trypsin, etc., it is possible to obtain a free form of theDNA-transfected cell, culture the cells or establish the cell line ofcultured cells. Furthermore, the DNA transgenic animal of the presentinvention can serve to identify cells capable of producing the proteinof the present invention, and to study in association with apoptosis,differentiation or propagation or on the mechanism of signaltransduction in these properties to inspect any abnormality therein.Thus, the DNA transgenic animal can provide an effective researchmaterial for the protein of the present invention and for investigationof its function and effect.

To develop a drug for the treatment of diseases associated with theprotein of the present invention, including the function inactive typeinadaptability to the protein of the present invention, using the DNAtransgenic animal of the present invention, an effective and rapidmethod for screening can be provided by using the method for inspectionand the method for quantification, etc. described above. It is alsopossible to investigate and develop a method for DNA therapy for thetreatment of diseases associated with the protein of the presentinvention, using the DNA transgenic animal of the present invention or avector capable of expressing the exogenous DNA of the present invention.

(8) Knockout Animal

The present invention provides a non-human mammal embryonic stem cellbearing the DNA of the present invention inactivated and a non-humanmammal deficient in expressing the DNA of the present invention.

Thus, the present invention provides:

-   (1) A non-human mammal embryonic stem cell in which the DNA of the    present invention is inactivated;-   (2) The embryonic stem cell according to (1), wherein the DNA is    inactivated by introducing a reporter gene (e.g., β-galactosidase    gene derived from Escherichia coli);-   (3) The embryonic stem cell according to (1), which is resistant to    neomycin;-   (4) The embryonic stem cell according to (1), wherein the non-human    mammal is a rodent;-   (5) The embryonic stem cell according to (4), wherein the rodent is    mouse;-   (6) A non-human mammal deficient in expressing the DNA of the    present invention, wherein the DNA is inactivated;-   (7) The non-human mammal according to (6), wherein the DNA is    inactivated by inserting a reporter gene (e.g., β-galactosidase    derived from Escherichia coli) therein and the reporter gene is    capable of being expressed under control of a promoter for the DNA    of the present invention;-   (8) The non-human mammal according to (6), which is a rodent;-   (9) The non-human mammal according to (8), wherein the rodent is    mouse; and,-   (10) A method of screening a compound or its salt that inhibits    (preferably inhibits) the promoter activity to the DNA of the    present invention, which comprises administering a test compound to    the mammal of (7) and detecting expression of the reporter gene.

The non-human mammal embryonic stem cell in which the DNA of the presentinvention is inactivated refers to a non-human mammal embryonic stemcell that suppresses the ability of the non-human mammal to express theDNA by artificially mutating the DNA of the present invention, or theDNA has no substantial ability to express the protein of the presentinvention (hereinafter sometimes referred to as the knockout DNA of thepresent invention) by substantially inactivating the activities of theprotein of the present invention encoded by the DNA (hereinafter merelyreferred to as ES cell).

As the non-human mammal, the same examples as described above apply.

Techniques for artificially mutating the DNA of the present inventioninclude deletion of a part or all of the DNA sequence and insertion ofor substitution with other DNA, by genetic engineering. By thesevariations, the knockout DNA of the present invention may be prepared,for example, by shifting the reading frame of a codon or by disruptingthe function of a promoter or exon.

Specifically, the non-human mammal embryonic stem cell in which the DNAof the present invention is inactivated (hereinafter merely referred toas the ES cell with the DNA of the present invention inactivated or theknockout ES cell of the present invention) can be obtained, for example,by isolating the DNA of the present invention that the desired non-humanmammal possesses, inserting a DNA fragment having a DNA sequenceconstructed by inserting a drug resistant gene such as a neomycinresistant gene or a hygromycin resistant gene, or a reporter gene suchas lacZ (β-galactosidase gene) or cat (chloramphenicol acetyltransferasegene), etc. into its exon site thereby to disable the functions of exon,or integrating to a chromosome of the target animal by, e.g., homologousrecombination, a DNA sequence that terminates gene transcription (e.g.,polyA additional signal, etc.) in the intron between exons, thusinhibiting the synthesis of complete messenger RNA and eventuallydestroying the gene (hereinafter simply referred to as a targetingvector). The thus-obtained ES cells to the southern hybridizationanalysis with a DNA sequence on or near the DNA of the present inventionas a probe, or to PCR analysis with a DNA sequence on the targetingvector and another DNA sequence near the DNA of the present inventionwhich is not included in the targeting vector as primers, to select theknockout ES cell of the present invention.

The parent ES cells to inactivate the DNA of the present invention byhomologous recombination, etc. may be of a strain already established asdescribed above, or may originally be established in accordance with amodification of the known method by Evans and Kaufma described above.For example, in the case of mouse ES cells, currently it is commonpractice to use ES cells of the 129 strain. However, since theirimmunological background is obscure, the C57BL/6 mouse or the BDF₁ mouse(F₁ hybrid between C57BL/6 and DBA/2), wherein the low ovum availabilityper C57BL/6 in the C57BL/6 mouse has been improved by crossing withDBA/2, may be preferably used, instead of obtaining a pure line of EScells with the clear immunological genetic background and for otherpurposes. The BDF₁ mouse is advantageous in that, when a pathologicmodel mouse is generated using ES cells obtained therefrom, the geneticbackground can be changed to that of the C57BL/6 mouse by back-crossingwith the C57BL/6 mouse, since its background is of the C57BL/6 mouse, aswell as being advantageous in that ovum availability per animal is highand ova are robust.

In establishing ES cells, blastocytes at 3.5 days after fertilizationare commonly used. In the present invention, embryos are preferablycollected at the 8-cell stage, after culturing until the blastocytestage, the embryos are used to efficiently obtain a large number ofearly stage embryos.

Although the ES cells used may be of either sex, male ES cells aregenerally more convenient for generation of a germ cell line chimera. Itis also desirable that sexes are identified as soon as possible to savepainstaking culture time.

Methods for sex identification of the ES cell include the method inwhich a gene in the sex-determining region on the Y-chromosome isamplified by the PCR process and detected. When this method is used, onecolony of ES cells (about 50 cells) is sufficient for sex-determinationanalysis, which karyotype analysis, for example G-banding method,requires about 10⁶ cells; therefore, the first selection of ES cells atthe early stage of culture can be based on sex identification, and malecells can be selected early, which saves a significant amount of time atthe early stage of culture.

Also, second selection can be achieved by, for example, confirmation ofthe number of chromosomes by the G-banding method. It is usuallydesirable that the chromosome number of the obtained ES cells be 100% ofthe normal number. However, when it is difficult to obtain the cellshaving the normal number of chromosomes due to physical operations, etc.in the cell establishment, it is desirable that the ES cell is againcloned to a normal cell (e.g., in a mouse cell having the number ofchromosomes being 2n=40) after knockout of the gene of the ES cells.

Although the embryonic stem cell line thus obtained shows a very highgrowth potential, it must be subcultured with great care, since it tendsto lose its ontogenic capability. For example, the embryonic stem cellline is cultured at about 37° C. in a carbon dioxide incubator(preferably 5% carbon dioxide and 95% air, or 5% oxygen, 5% carbondioxide and 90% air) in the presence of LIF (1 to 10000 U/ml) onappropriate feeder cells such as STO fibroblasts, treated with atrypsin/EDTA solution (normally 0.001 to 0.5% trypsin/0.1 to about 5 mMEDTA, preferably about 0.1% trypsin/1 mM EDTA) at the time of passage toobtain separate single cells, which are then plated on freshly preparedfeeder cells. This passage is normally conducted every 1 to 3 days; itis desirable that cells be observed at the passage and cells found to bemorphologically abnormal in culture, if any, be abandoned.

Where ES cells are allowed to reach a high density in mono-layers or toform cell aggregates in suspension under appropriate conditions, it ispossible to differentiate the ES cells to various cell types, forexample, pariental and visceral muscles, cardiac muscle or the like[Nature, 292, 154, 1981; Proc. Natl. Acad. Sci. U.S.A., 78, 7634, 1981;Journal of Embryology Experimental Morphology, 87, 27, 1985]. The cellsdeficient in expression of the DNA of the present invention, which areobtained from the differentiated ES cells of the present invention, areuseful for studying the function of the protein of the present inventioncytologically.

The non-human mammal deficient in expression of the DNA of the presentinvention can be identified from a normal animal by measuring the mRNAlevel in the subject animal by a publicly known method, and indirectlycomparing the degrees of expression.

As the non-human mammal, the same examples supra apply.

With respect to the non-human mammal deficient in expression of the DNAof the present invention, the DNA of the present invention can be madeknockout by transfecting a targeting vector, prepared as describedabove, to mouse embryonic stem cells or mouse oocytes, and conductinghomologous recombination in which a targeting vector DNA sequence,wherein the DNA of the present invention is inactivated by thetransfection, is replaced with the DNA of the present invention on achromosome of a mouse embryonic stem cell or mouse embryo.

The knockout cells with the disrupted DNA of the present invention canbe identified by the southern hybridization analysis using as a probe aDNA fragment at the site proximal to the DNA of the present invention,or by the PCR analysis using as primers a DNA sequence on the targetingvector and another DNA sequence at the proximal region of other than theDNA of the present invention derived from mouse used in the targetingvector. When non-human mammal stem cells are used, a cell line whereinthe DNA of the present invention is inactivated by homologousrecombination is cloned; the resulting clones are injected to, e.g., anon-human mammalian embryo or blastocyst, at an appropriate stage suchas the 8-cell stage. The resulting chimeric embryos are transplanted tothe uterus of the pseudopregnant non-human mammal. The resulting animalis a chimeric animal constructed with both cells having the normal locusof the DNA of the present invention and those having an artificiallymutated locus of the DNA of the present invention.

When some germ cells of the chimeric animal have a mutated locus of theDNA of the present invention, an individual, which entire tissue iscomposed of cells having a mutated locus of the DNA of the presentinvention can be selected from a series of offspring obtained bycrossing between such a chimeric animal and a normal animal, e.g., bycoat color identification, etc. The individuals thus obtained arenormally deficient in heterozygous expression of the protein of thepresent invention. The individuals deficient in homozygous expression ofthe protein of the present invention can be obtained from offspring ofthe intercross between those deficient in heterozygous expression of theprotein of the present invention.

When an oocyte is used, a DNA solution may be injected, e.g., into theprenucleus by microinjection thereby to obtain a transgenic non-humanmammal having a targeting vector introduced in its chromosome. From suchtransgenic non-human mammals, those having a mutation at the locus ofthe DNA of the present invention can be obtained by selection based onhomologous recombination.

As described above, the individuals in which the DNA of the presentinvention is rendered knockout permit passage rearing under ordinaryrearing conditions, after the individuals obtained by their crossinghave proven to have been knockout.

Furthermore, the genital system may be obtained and retained byconventional methods. That is, by crossing male and female animals eachhaving the inactivated DNA, homozygote animals having the inactivatedDNA in both loci can be obtained. The homozygotes thus obtained may bereared so that one normal animal and two or more homozygotes areproduced from a mother animal to efficiently obtain such homozygotes. Bycrossing male and female heterozygotes, homozygotes and heterozygoteshaving the inactivated DNA are proliferated and passaged.

The non-human mammal embryonic stem cell, in which the DNA of thepresent invention is inactivated, is very useful for preparing anon-human mammal deficient in expression of the DNA of the presentinvention.

Since the non-human mammal, in which the DNA of the present invention isinactivated, lacks various biological activities derived from theprotein of the present invention, such an animal can be a disease modelsuspected of inactivated biological activities of the protein of thepresent invention and thus, offers an effective study to investigate thecauses for and therapy for these diseases.

(8a) Method of Screening a Compound Having a Therapeutic and/orPreventive Effect on Diseases Caused by Deficiency, Damages, Etc. of theDNA of the Present Invention

The non-human mammal deficient in expression of the DNA of the presentinvention can be employed for screening of a compound having atherapeutic and/or preventive effect on diseases caused by deficiency,damages, etc. of the DNA of the present invention.

That is, the present invention provides a method of screening a compoundhaving a therapeutic and/or preventive effect on diseases caused bydeficiency, damages, etc. of the DNA of the present invention, forexample, an effect of sensitizing insulin, or a therapeutic and/orpreventive effect on obesity, hyperlipemia, arteriosclerosis,hypertension or heart disease, which comprises administering a testcompound to a non-human mammal deficient in expression of the DNA of thepresent invention and, observing and measuring a change occurred in theanimal.

As the non-human mammal deficient in expression of the DNA of thepresent invention, which can be employed for the screening method, thesame examples as given hereinabove apply.

Examples of the test compound include peptides, proteins, non-peptidecompounds, synthetic compounds, fermentation products, cell extracts,plant extracts, animal tissue extracts, blood plasma, etc. Thesecompounds may be novel compounds or publicly known compounds.

Specifically, the non-human mammal deficient in expression of the DNA ofthe present invention is treated with a test compound, comparison ismade with an intact animal for control and a change in each organ,tissue, disease conditions, etc. of the animal is used as an indicatorto assess the therapeutic and/or preventive effects of the testcompound.

For treating an animal to be tested with a test compound, for example,oral administration, intravenous injection, etc. are applied, and thetreatment can be appropriately selected depending on conditions of thetest animal, properties of the test compound, etc. Furthermore, a doseof the test compound to be administered can be appropriately selecteddepending on the administration route, nature of the test compound, etc.

For screening of the compound having an effect of sensitizing insulin,or a preventive and/or therapeutic effect on, e.g., obesity,hyperlipemia, arteriosclerosis, hypertension or heart disease, a testcompound is given to a non-human mammal enhanced in expression of theDNA of the present invention, which suffers from the pathologicalconditions described above, during fasting, after feeding, or before andafter insulin administration. Then, the level of insulin, glucose orfree fatty acid contained in blood is observed with passage of time, ascompared to the group added with no test compound.

When a test compound is given to a test animal in the screening methodabove and the aforesaid disease conditions of the test animal areimproved at least by about 10%, preferably at least by about 30%, morepreferably at least by about 50%, the test compound can be selected as acompound having the therapeutic and/or preventive effects on thediseases described above.

The compound obtained using the above screening method is a compoundselected from the test compounds described above and exhibits apreventive and/or therapeutic effect on the diseases caused bydeficiencies, damages, etc. of the protein of the present invention.Therefore, the compound can be employed as a safe and low toxic drugsuch as an agent for the prevention and/or treatment of these diseases.Furthermore, compounds derived from the compound obtained by thescreening described above may also be used as well.

The compound obtained by the screening method above may form salts, andmay be used in the form of salts with physiologically acceptable acids(e.g., inorganic acids, organic acids, etc.) or bases (e.g., alkalimetal salts), preferably in the form of physiologically acceptable acidaddition salts. Examples of such salts are salts with inorganic acids(e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuricacid, etc.), salts with organic acids (e.g., acetic acid, formic acid,propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid,citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonicacid, benzenesulfonic acid, etc.) and the like.

A pharmaceutical which comprises the compound obtained by the abovescreening method or salts thereof can be manufactured in a mannersimilar to the method for preparing the pharmaceutical which comprisesthe protein of the present invention described hereinabove.

As the pharmaceutical preparation thus obtained is safe and low toxic,it can be administered to human or mammal (e.g., rat, mouse, guinea pig,rabbit, sheep, swine, bovine, horse, cat, dog, monkey, etc.).

The dose of the compound or its salt may vary depending upon targetdisease, subject to be administered, route of administration, etc. Forexample, when the compound is orally administered, the compound isgenerally administered to an adult patient (as 60 kg body weight) withinsulin sensitization in a daily dose of about 0.1 to about 100 mg,preferably about 1.0 to about 50 mg and more preferably about 1.0 toabout 20 mg. In parenteral administration, a single dose of the compoundmay vary depending upon subject to be administered, target disease, etc.When the compound is administered to an adult (as 60 kg body weight) inthe form of an injectable preparation, it is advantageous to administerthe compound intravenously to the patient with insulin sensitization ina daily dose of about 0.01 to about 30 mg, preferably about 0.1 to about20 mg, and more preferably about 0.1 to about 10 mg. For other animalspecies, the corresponding dose as converted per 60 kg weight can beadministered.

(8b) Method of Screening a Compound that Inhibits the Activity of aPromoter to the DNA of the Present Invention

The present invention provides a method of screening a compound or itssalts that inhibit the activity of a promoter to the DNA of the presentinvention, which comprises administering a test compound to a non-humanmammal deficient in expression of the DNA of the present invention anddetecting the expression of the reporter gene.

In the screening method described above, an animal in which the DNA ofthe present invention is inactivated by introducing a reporter gene andthe reporter gene is expressed under control of a promoter to the DNA ofthe present invention is used as the non-human mammal deficient inexpression of the DNA of the present invention, which is selected fromthe aforesaid non-human mammals deficient in expression of the DNA ofthe present invention.

The same examples of the test compound apply to specific compounds usedfor the screening.

As the reporter gene, the same specific examples apply to this screeningmethod. Preferably, there are used β-galactosidase (lacZ), solublealkaline phosphatase gene, luciferase gene and the like.

Since the reporter gene is present under control of a promoter to theDNA of the present invention in the non-human mammal deficient inexpression of the DNA of the present invention wherein the DNA of thepresent invention is replaced by the reporter gene, the activity of thepromoter can be detected by tracing the expression of a substanceencoded by the reporter gene.

When a part of the DNA region encoding the protein of the presentinvention is substituted with, e.g., β-galactosidase gene (lacZ) derivedfrom Escherichia coli, β-galactosidase is expressed in a tissue wherethe protein of the present invention should originally be expressed,instead of the protein of the present invention. Thus, the state ofexpression of the protein of the present invention can be readilyobserved in vivo of an animal by staining with a reagent, e.g.,5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal) which issubstrate for β-galactosidase. Specifically, a mouse deficient in theprotein of the present invention, or its tissue section is fixed withglutaraldehyde, etc. After washing with phosphate buffered saline (PBS),the system is reacted with a staining solution containing X-gal at roomtemperature or about 37° C. for approximately 30 minutes to an hour.After the β-galactosidase reaction is terminated by washing the tissuepreparation with 1 mM EDTA/PBS solution, the color formed is observed.Alternatively, mRNA encoding lacZ may be detected in a conventionalmanner.

The compound or its salt obtained using the screening method describedabove is the compound that is selected from the test compounds describedabove and that inhibits the promoter activity to the DNA of the presentinvention.

The compound obtained by the screening method above may form salts, andmay be used in the form of salts with physiologically acceptable acids(e.g., inorganic acids, etc.) or bases (e.g., organic acids, etc.) orthe like, especially in the form of physiologically acceptable acidaddition salts. Examples of such salts are salts with inorganic acids(e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuricacid, etc.), salts with organic acids (e.g., acetic acid, formic acid,propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid,citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonicacid, benzenesulfonic acid, etc.) and the like.

The compound or its salt that inhibits the promoter activity to the DNAof the present invention can regulate the expression of the protein ofthe present invention and regulate the function of the protein. Thus,the compound or its salt is useful as an insulin-sensitizing agent, oran agent for the prevention and/or treatment of obesity, hyperlipemia,arteriosclerosis, hypertension or heart disease.

In addition, compounds derived from the compound obtained by thescreening described above may also be used as well.

A pharmaceutical which comprises the compound obtained by the abovescreening method or its salt can be manufactured in a manner similar tothe method for preparing the pharmaceutical which comprises the proteinof the present invention described hereinabove.

As the pharmaceutical preparation thus obtained is safe and low toxic,it can be administered to human or mammal (e.g., rat, mouse, guinea pig,rabbit, sheep, swine, bovine, horse, cat, dog, monkey, etc.).

A dose of the compound or its salt may vary depending on target disease,subject to be administered, route for administration, etc.; when thecompound that inhibits the promoter activity to the DNA of the presentinvention is orally administered to an adult (as 60 kg body weight), thecompound is administered to the patient with insulin sensitizationnormally in a daily dose of about 0.1 to about 100 mg, preferably about1.0 to about 50 mg and more preferably about 1.0 to about 20 mg. Inparenteral administration, a single dose of the compound variesdepending on subject to be administered, target disease, etc. but whenthe compound of inhibiting the promoter activity to the DNA of thepresent invention is administered to an adult (as 60 kg body weight) inthe form of injectable preparation, it is advantageous to administer thecompound intravenously to the patient with insulin sensitization in adaily dose of about 0.01 to about 30 mg, preferably about 0.1 to about20 mg and more preferably about 0.1 to about 10 mg. For other animalspecies, the corresponding dose as converted per 60 kg weight can beadministered.

As stated above, the non-human mammal deficient in expression of the DNAof the present invention is extremely useful for screening the compoundor its salt that inhibits the promoter activity to the DNA of thepresent invention and, can greatly contribute to elucidation of causesfor various diseases suspected of deficiency in expression of the DNA ofthe present invention and for the development of preventive and/ortherapeutic agent for these diseases.

Also, a so-called transgenic animal (gene transferred animal) can beprepared by using a DNA containing the promoter region of the protein ofthe present invention, ligating genes encoding various proteins at thedownstream and injecting the same into oocyte of an animal. It is thuspossible to synthesize the protein therein specifically and study itsactivity in vivo. When an appropriate reporter gene is ligated to thepromoter site described above and a cell line that expresses the gene isestablished, the resulting system can be utilized as the search systemfor a low molecular compound having the action of specificallyinhibiting the in vivo productivity of the protein of the presentinvention itself.

(9) Determination of a Ligand to the Protein of the Present Invention

The protein of the present invention, its partial peptide, or a saltthereof is useful as a reagent for searching or determining a ligand tothe protein of the present invention or its salt.

The ligand to the protein of the present invention is determined bycontacting the protein of the present invention or its salt or thepartial peptide of the protein of the present invention or its salt witha test compound.

Examples of the test compound used include tissue extracts from mammal(e.g., human, mouse, rat, swine, bovine, sheep, monkey, etc.), cellculture supernatant, proteins or partial peptides expressed asrecombinants by random peptide library or mammal cDNA library, and thelike. For example, the tissue extract, cell culture supernatant or thelike is added to the protein of the present invention and the mixture isfractionated while assaying the cell stimulating activity so that asingle ligand can eventually be obtained. Also, the ligand can beobtained by identifying a binding protein using the binding activity ofa random peptide or cDNA library-expressing bacterium or bacteriophageto the protein of the present invention as an indicator, or identifyinga binding protein by the yeast two-hybrid technique using a randompeptide or cDNA library and yeast expressing the protein, and assayingthe cell stimulating activity, etc. from these binding proteins.

Specifically, the method of determining a ligand in the presentinvention is a method of determining a compound (e.g., a peptide,protein, non-peptide compound, synthetic compound, fermentation product,etc.) or its salt, which binds to the protein of the present inventionto induce the cell stimulating activity (the activity of promoting orsuppressing, e.g., phosphorylation of the protein of the presentinvention, phosphorylation of ERK, production of TNF-α, intracellularCa²⁺ release, change in cell membrane potential, intracellular proteinphosphorylation, pH reduction, etc.), by using the protein of thepresent invention, its partial peptide, or a salt thereof, or byconstructing the expression system of recombinant protein and using thereceptor binding assay system using the expression system.

The method of determining the ligand in the present invention comprisesassaying the amount of a test compound bound to the protein of thepresent invention or its partial peptide or the cell stimulatingactivity, when the protein or its partial peptide is brought in contactwith the test compound.

More specifically, the present invention provides:

-   -   (1) a method of determining a ligand to the protein of the        present invention or its salt, which comprises purifying a test        compound bound to the protein of the present invention or its        salt or the partial peptide of the present invention or its        salt, when the tissue extract from a mammal (e.g., human, mouse,        rat, swine, bovine, sheep, monkey, etc.), serum, cell culture        supernatant, etc. is brought in contact with the protein or its        salt or the partial peptide or its salt;    -   (2) a method of determining a ligand to the protein of the        present invention or its salt, which comprises purifying a test        compound bound to the protein of the present invention or its        partial peptide, when the tissue extract from a mammal (e.g.,        human, mouse, rat, swine, bovine, sheep, monkey, etc.), serum,        cell culture supernatant, etc. is brought in contact with a cell        containing the protein or a membrane fraction of the cell;    -   (3) a method of determining a ligand to the protein of the        present invention, which comprises purifying a test compound        bound to the protein of the present invention or its partial        peptide, when the tissue extract from a mammal (e.g., human,        mouse, rat, swine, bovine, sheep, monkey, etc.), serum, cell        culture supernatant, etc. is brought in contact with a protein        expressed on a cell membrane by culturing a transformant        containing a DNA encoding the protein of the present invention;    -   (4) a method of determining a ligand to the protein of the        present invention, which comprises contacting a bacterium or        bacteriophage expressing a random peptide or cDNA library on the        surface with the protein of the present invention and        identifying the amino acid sequence of a peptide binding to the        protein;    -   (5) a method of determining a ligand to the protein of the        present invention, which comprises identifying the amino acid        sequence of a peptide binding to the protein by the yeast        two-hybrid technique using a random peptide or cDNA library and        yeast expressing the protein of the present invention;    -   (6) a method of determining a ligand to the protein of the        present invention or its salt, which comprises assaying the        protein-mediated cell stimulating activity (the activity of        promoting or suppressing, e.g., phosphorylation of the protein        of the present invention, phosphorylation of ERK, production of        TNF-α, intracellular Ca²⁺ release, change in cell membrane        potential, intracellular protein phosphorylation, pH reduction,        etc.) in the case where a test compound wherein a binding        activity has been found is brought in contact with a cell        containing the protein of the present invention; and,    -   (7) a method of determining a ligand to the protein of the        present invention or its salt, which comprises assaying the        protein-mediated cell stimulating activity (the activity of        promoting or suppressing, e.g., phosphorylation of the protein        of the present invention, phosphorylation of ERK, production of        TNF-α, intracellular Ca²⁺ release, change in cell membrane        potential, intracellular protein phosphorylation, pH reduction,        etc.) in the case where a protein expressed on a cell membrane        by culturing a transformant containing a DNA encoding the        protein of the present invention. a test compound wherein a        binding activity has been found is brought in contact with a        cell containing the protein of the present invention

It is particularly preferred to perform the tests (1) to (5) describedabove, thereby to confirm that the test compound can bind to the proteinof the present invention, followed by the tests (6) and (7) describedabove.

Any protein is usable as the protein for the ligand determination, solong as it contains the protein of the present invention or the partialpeptide of the present invention. However, the protein abundantlyexpressed using animal cells is appropriate.

To manufacture the protein of the present invention, the expressionmethods described above are used and preferably, the protein ismanufactured by expressing a DNA encoding the protein in mammalian orinsect cells. For the DNA fragment encoding the objective proteinregion, normally the complementary DNA, but not necessarily limitedthereto, is employed. For example, the gene fragments or synthetic DNAmay also be used. To introduce a DNA fragment encoding the protein ofthe present invention into host animal cells and efficiently express theDNA there, it is preferred to insert the DNA fragment downstream of apolyhedorin promoter of nuclear polyhedrosis virus (NPV) belonging tobaculovirus hosted by insects, SV40-derived promoter, retroviruspromoter, metallothionein promoter, human heat shock promoter,cytomegalovirus promoter, SRα promoter, etc. The quantity and quality ofthe expressed receptor are examined by publicly known methods, forexample, the method described in the literature (J. Biol. Chem., 267,19555-19559, 1992).

Therefore, in the ligand determination methods of the present invention,the material containing the protein of the present invention, itspartial peptide, or a salt thereof, may be a protein purified bypublicly known methods, its partial peptide, or a salt thereof, or acell containing the protein, or a membrane fraction of the cell.

In the ligand determination methods of the present invention, when cellscontaining the protein of the present invention are used, the cells maybe fixed with glutaraldehyde, formalin, etc. The cells can be fixed bypublicly known methods.

Cells containing the protein of the present invention refer to hostcells that have expressed the protein of the present invention, whichhost cells include Escherichia coli, Bacillus subtilis, yeast, insectcells, animal cells, etc.

The cell membrane fraction is a fraction abundant in cell membraneobtained by cell disruption and subsequent fractionation by a publiclyknown method. Useful cell disruption methods include cell squashingusing a Potter-Elvehjem homogenizer, disruption using a Waring blenderor Polytron (manufactured by Kinematica, Inc.), disruption byultrasonication, disruption by cell spraying via a thin nozzle underincreased pressure using a French press, and the like. Cell membranefractionation is effected mainly by fractionation using a centrifugalforce, such as centrifugation for fractionation, density gradientcentrifugation, etc. For example, cell disruption fluid is centrifugedat a low rate (500 rpm to 3,000 rpm) for a short period of time(normally about 1 to 10 minutes), the resulting supernatant is thencentrifuged at a higher rate (15,000 to 30,000 rpm) normally for 30minutes to 2 hours. The precipitate thus obtained is used as themembrane fraction. The membrane fraction is abundant in the proteinexpressed and membrane components such as cell-derived phospholipids,membrane proteins, etc.

The amount of the protein in the cells containing the protein and in themembrane fraction is preferably 10³ to 10⁸ molecules per cell, morepreferably 10⁵ to 10⁷ molecules per cell. As the level of expression isenhanced, the ligand binding activity per unit of the membrane fraction(specific activity) increases so that not only a highly sensitivescreening system can be constructed but also large quantities of samplescan be assayed with the same lot.

The ligand to the protein of the present invention or its salt isdetermined by the following procedures. First, a preparation of theprotein of the present invention is prepared by suspending a cellcontaining the protein of the present invention or a membrane fractionof the cell in a buffer appropriate for use in the determination method.Any buffer may be used if it does not interfere with the ligand-proteinbinding, such buffers including a phosphate buffer, a Tris-HCl buffer,etc., having a pH of 4 to 10 (preferably a pH of 6 to 8). For thepurpose of minimizing non-specific binding, a surfactant such as CHAPS,Tween-80™ (Kao-Atlas Inc.), digitonin, deoxycholate, etc., or variousproteins such as bovine serum albumin, gelatin or the like, mayoptionally be added to the buffer. Further for the purpose ofsuppressing the degradation of the receptor or ligand by protease, aprotease inhibitor such as PMSF, leupeptin, E-64 (manufactured byPeptide Institute, Inc.), pepstatin, etc. may also be added. A givenamount (5,000 cpm to 500,000 cpm) of the test compound labeled with[³H], [¹²⁵I], [¹⁴C], [³⁵S] or the like is added to 0.01 ml to 10 ml ofthe receptor solution. To determine the amount of non-specific binding(NSB), a reaction tube containing an unlabeled test compound in a largeexcess is prepared as well. The reaction is carried out approximately at0 to 50° C., preferably about 4 to 37° C. for about 20 minutes to about24 hours, preferably about 30 minutes to about 3 hours. After completionof the reaction, the reaction mixture is filtrated through glass fiberfilter paper, etc. and rinsed with an appropriate amount of the samebuffer. The residual radioactivity in the glass fiber filter paper isthen measured by means of a liquid scintillation counter or a γ-counter.A test compound exceeding 0 cpm in count obtained by subtractingnonspecific binding (NSB) from the total binding (B) (13 minus NSB) canbe selected as a ligand to the protein of the present invention or itssalt.

The method (6) or (7) above for determining a ligand to the protein ofthe present invention or its salt can be performed as follows. Theprotein-mediated cell-stimulating activity (the activity of promoting orsuppressing, e.g., phosphorylation of the protein of the presentinvention, phosphorylation of ERK, production of TNF-α, intracellularCa²⁺ release, change in cell membrane potential, intracellular proteinphosphorylation, pH reduction, etc.) can be assayed by a publicly knownmethod, or using an assay kit commercially available. Specifically,cells containing the protein are cultured on a multiwell plate, etc.Prior to the ligand determination, the medium is replaced with freshmedium or with an appropriate non-cytotoxic buffer, followed byincubation for a given period of time in the presence of a testcompound, etc. Subsequently, the cells are extracted or the supernatantis recovered and the product formed is quantified by appropriateprocedures. Where it is difficult to detect the production of anindicator substance for the cell stimulating activity due to a degradingenzyme contained in the cells, an inhibitor against such a degradingenzyme may be added prior to the assay.

The kit of the present invention for determining a ligand that binds tothe protein of the present invention or its salt comprises the proteinof the present invention or its salt, the partial peptide of the presentinvention or its salt, a cell containing the protein of the presentinvention, or a membrane fraction of the cell containing the protein ofthe present invention.

Examples of the ligand determination kit of the present invention aregiven below.

1. Reagent for Ligand Determination

(1) Assay Buffer and Wash Buffer

Hanks' Balanced Salt Solution (manufactured by Gibco Co.) supplementedwith 0.05% bovine serum albumin (Sigma Co.).

The solution is sterilized by filtration through a 0.45 μm filter andstored at 4° C. Alternatively, the solution may be prepared at use.

(2) Preparation of the Protein of the Present Invention

CHO cells on which the protein of the present invention has beenexpressed are subcultured in a 12-well plate at the rate of 5×10⁵cells/well and then cultured at 37° C. under 5% CO₂ and 95% air for 2days.

(3) Labeled Test Compound

A compound labeled with commercially available [³H], [¹²⁵I], [¹⁴C],[³⁵S], etc., or a compound labeled by appropriate methods.

An aqueous solution of the compound is stored at 4° C. or −20° C. Thesolution is diluted to 1 μM with an assay buffer at use. A sparinglywater-soluble test compound is dissolved in dimethylformamide, DMSO,methanol, etc.

(4) Unlabeled Test Compound

Unlabeled form of the same test compound is prepared in a concentration100 to 1,000-fold higher than that of the labeled compound.

2. Assay Procedures

(1) CHO cells capable of expressing the protein of the present inventionare cultured in a 12-well culture plate. After washing twice with 1 mlof assay buffer, 490 μl of the assay buffer is added to each well.

(2) After 5 μl of a labeled test compound is added, the resultingmixture is incubated at room temperature for an hour. To determine thenon-specific binding, 5 μl of unlabeled compound is added to the system.

(3) The reaction mixture is removed and the wells are washed 3 timeswith 1 ml of wash buffer. The labeled test compound bound to the cellsis dissolved in 0.2N NaOH-1% SDS and then mixed with 4 ml of liquidscintillator A (manufactured by Wako Pure Chemical Industries, Ltd.).

(4) The radioactivity is measured using a liquid scintillation counter(manufactured by Beckman Co.).

In the specification, the codes of bases and amino acids are denoted inaccordance with the IUPAC-IUB Commission on Biochemical Nomenclature orby the common codes in the art, examples of which are shown below. Foramino acids that may have the optical isomer, L form is presented unlessotherwise indicated.

-   -   DNA: deoxyribonucleic acid    -   cDNA: complementary deoxyribonucleic acid    -   A: adenine    -   T: thymine    -   G: guanine    -   C: cytosine    -   RNA: ribonucleic acid    -   mRNA: messenger ribonucleic acid    -   dATP: deoxyadenosine triphosphate    -   dTTP: deoxythymidine triphosphate    -   dGTP: deoxyguanosine triphosphate    -   dCTP: deoxycytidine triphosphate    -   ATP: adenosine triphosphate    -   EDTA: ethylenediaminetetraacetic acid    -   SDS: sodium dodecyl sulfate    -   Gly: glycine    -   Ala: alanine    -   Val: valine    -   Leu: leucine    -   Ile: isoleucine    -   Ser: serine    -   Thr: threonine    -   Cys: cysteine    -   Met: methionine    -   Glu: glutamic acid    -   Asp: aspartic acid    -   Lys: lysine    -   Arg: arginine    -   His: histidine    -   Phe: phenylalanine    -   Tyr: tyrosine    -   Trp: tryptophan    -   Pro: proline    -   Asn: asparagine    -   Gln: glutamine    -   pGlu: pyroglutamic acid    -   Sec: selenocysteine

Substituents, protecting groups and reagents frequently used in thisspecification are presented as the codes below.

-   -   Me: methyl group    -   Et: ethyl group    -   Bu: butyl group    -   Ph: phenyl group    -   TC: thiazolidine-4(R)-carboxamide group    -   Tos: p-toluenesulfonyl    -   CHO: formyl    -   Bzl: benzyl    -   Cl₂-Bzl: 2,6-dichlorobenzyl    -   Bom: benzyloxyymethyl    -   Z: benzyloxycarbonyl    -   Cl-Z: 2-chlorobenzyloxycarbonyl    -   Br-Z: 2-bromobenzyl oxycarbonyl    -   Boc: t-butoxycarbonyl    -   DNP: dinitrophenol    -   Trt: trityl    -   Bum: t-butoxymethyl    -   Fmoc: N-9-fluorenyl methoxycarbonyl    -   HOBt: 1-hydroxybenztriazole    -   HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine    -   HONB: 1-hydroxy-5-norbornene-2,3-dicarboxyimide    -   DCC: N,N′-dichlorohexylcarbodiimide        The sequence identification numbers in the sequence listing of        the specification indicate the following sequences.        [SEQ ID NO: 1]

This shows the amino acid sequence of human TREM-2

[SEQ ID NO: 2]

This shows the amino acid sequence of mouse TREM-2

[SEQ ID NO: 3]

This shows the base sequence of DNA encoding human TREM-2 having theamino acid sequence represented by SEQ ID NO: 1.

[SEQ ID NO: 4]

This shows the base sequence of DNA encoding human TREM-2 having theamino acid sequence represented by SEQ ID NO: 2.

[SEQ ID NO: 5]

This shows the base sequence of the primer used in EXAMPLE 1.

[SEQ ID NO: 6]

This shows the base sequence of the primer used in EXAMPLE 1.

[SEQ ID NO: 7]

This shows the base sequence of the primer used in EXAMPLE 2.

[SEQ ID NO: 8]

This shows the base sequence of the primer used in EXAMPLE 2.

[SEQ ID NO: 9]

This shows the base sequence of the primer used in EXAMPLE 2 and EXAMPLE3.

[SEQ ID NO: 10]

This shows the base sequence of the primer used in EXAMPLE 2 and EXAMPLE3.

[SEQ ID NO: 11]

This shows the base sequence of the primer used in EXAMPLE 6.

[SEQ ID NO: 12]

This shows the base sequence of the primer used in EXAMPLE 6.

[SEQ ID NO: 13]

This shows the base sequence of the primer used in EXAMPLE 6.

[SEQ ID NO: 14]

This shows the base sequence of the primer used in EXAMPLE 6.

[SEQ ID NO: 15]

This shows the base sequence of the primer used in EXAMPLE 6.

[SEQ ID NO: 16]

This shows the base sequence of the primer used in EXAMPLE 6.

Hereinafter, the present invention will be described more specificallywith reference to EXAMPLES, but is not intended to limit the presentinvention thereto.

EXAMPLE 1

Search of Expression-Shifted Gene

Epididymis adipose tissues were excised from KKA^(y) mouse (14 weeksold, male) and C57BL/6 mouse (14 weeks old, male), homogenized in ISOGENreagent (Wako Pure Chemical Industries, Ltd.) and extracted withchloroform followed by isopropanol precipitation to extract total RNAsfrom the respective tissues. From the total RNAs, poly (A)⁺ RNAs werefurther purified with Oligotex dT30 (Takara Bio Inc.). Using 2 μg ofthese poly (A)⁺ RNAs as the starting material, cDNA was synthesized byReverse Transcriptase (Superscript RTII; Invitrogen). Using PCR-SelectcDNA Subtraction Kit (Clontech), the group of cDNA, which expressionlevel increased or decreased in the KKA^(y) mouse adipose tissue, wasselectively amplified as PCR fragment. After ligation of the amplifiedcDNA group to cloning vector pT7 Blue-T, cloning was effected bytransforming Escherichia coli DH5α. The respective insertion sequenceswere amplified by vector sequences M13 primer P7 (SEQ ID NO: 5) and M13primer P8 (SEQ ID NO: 6) (both Toyobo). The resulting PCR fragments werespotted onto glass slides to prepare microarrays. Poly (A)⁺ RNA derivedfrom the adipose tissue in each mouse was fluorescence-labeled with Cy5or Cy3 by random priming to prepare a detection probe, which washybridized to microarray for 15 hours. The gene groups showing a markeddifference in the expression level were identified. When compared to theC57BL/6 mouse expression gene for control, 29 genes were identified asgenes with increased expression in KKA^(y) by 4 times or more; on theother hand, 69 genes were identified as genes with decreased expressionby 4 times or less. Mouse TREM-2 was identified to be a gene withincreased expression by 5.32 times in the gene group with increasedexpression.

EXAMPLE 2

Cloning of TREM-2 from Adipocytes

In order to acquire experimental materials for confirmation ofexpression shift of TREM-2 and for its function analysis, thefull-length coding region cDNA of TREM-2 was cloned by PCR from cDNAlibraries of human adipocyte and mouse 3T3-L1 adipocyte.

For cloning of human TREM-2, the following sequences were used asprimers. 5′-ATGGAGCCTCTCCGGCTGCTCATC-3′ (SEQ ID NO: 7)5′-TCACGTGTCTCTCAGCCCTGGCAG-3′ (SEQ ID NO: 8)

Using Advantage-2 cDNA PCR Kit (Clontech), the reaction was carried outin 35 cycles with one cycle set at 98° C. for 20 seconds and 68° C. for1 minute and 30 seconds.

For cloning of mouse TREM-2, the following sequences were used asprimers. 5′-ATGGGACCTCTCCACCAGTTTCTCCTG-3′ (SEQ ID NO: 9)5′-TCACGTACCTCCGGGTCCAGTGAG-3′ (SEQ ID NO: 10)

Using Pfu Turbo DNA polymerase (Stratagene), the reaction was carriedout in 35 cycles with one cycle set at 95° C. for 20 seconds, 65° C. for40 seconds and 72° C. for 1 minute.

The cDNA fragment obtained with human TREM-2 was used as it was. On theother hand, the cDNA fragment obtained with mouse TREM-2 was reacted onTAKARA Ex-Taq (Takara Bio Inc.) at 72° C. for 10 minutes to add A at theboth 3′ ends of the PCR fragment. Thereafter, the cDNA fragments werecloned to pCR2.1 vector (Invitrogen), respectively, followed by DNAsequencing. TREM-2 isolated from human adipocyte coincided with knownhuman TREM-2 (AF213457). On the other hand, 3 cDNAs of TREM-2a(AY024348), TREM-2b (AY024349) and TREM-2c (AF213458) are present asknown sequences for mouse TREM-2; in TREM-2 acquired from 3T3-L1adipocyte, 6 clones obtained were all coincident with TREM-2a.

EXAMPLE 3

Confirmation of Expression Shift by RT-PCR

RNAs extracted from epididymis adipose tissues of KKA^(y) mouse (14weeks old, male) and C57BL/6 mouse (14 weeks old, male) were subjectedto RT-PCR. Using an Oligo-dT-adapter primer (Takara Bio Inc.), cDNA wassynthesized with AMV (Avian Myeloblastosis Virus)-derived reversetranscriptase (Takara Bio Inc.) from 0.5 μg of total RNA extracted fromeach tissue. PCR was then conducted using primers (SEQ ID NO: 9 and SEQID NO: 10) for mouse TREM-2 cloning. Using Advantage-2 cDNA PCR Kit(Clontech), the reaction was carried out in 25 cycles with one cycle setat 98° C. for 20 seconds and 68° C. for 1 minute and 30 seconds. Bydetecting the reaction product on agarose gel electrophoresis, it couldbe confirmed that there was a difference in the TREM-2 expression levelin KKA^(y) mouse adipose tissue by 5 times or more than the TREM-2expression level in C57BL/6 mouse, as in the results with the microarrayin EXAMPLE 1.

EXAMPLE 4

Production of Anti-Mouse TREM-2 Antibody and Analysis of TREM-2Expression in Mouse Adipose Tissue on a Protein Level

A rabbit was immunized with a peptide constructed from 133 Leu to 147Ser in mouse TREM-2 sequence [SEQ ID NO: 2] as an antigen to acquireanti-TREM-2 polyclonal antibody. Using this antibody, 20 μg each of thehomogenates from the excised epididymis adipose tissues of KKA^(y) mouseand C57BL/6 mouse was analyzed by western blotting. The band of TREM-2was detected from the KKA^(y) mouse-derived tissue, whereas any band wasnot detected from the C57BL/6 mouse-derived tissue.

EXAMPLE 5

Distribution of TREM-2-Expressed Tissues in Mice

Total RNAs were extracted from the epididymis adipose tissue, mesentericadipose tissue, skeletal muscle, liver, testis, spleen, brain and kidneyof KKA^(y) mouse and C57BL/6 mouse, respectively. Distribution ofTREM-2-expressed tissues was then analyzed by RT-PCR described inEXAMPLE 3. A marked expression was noted in the epididymis adiposetissue and mesenteric adipose tissue in KKA^(y) mouse. On the otherhand, expression was barely noted in tissues other than these adiposetissues.

EXAMPLE 6

Quantification of TREM-2 in Diabetes Model Mouse

(1) Quantitative RT-PCR

The copy number of TREM-2 mRNA per 1 ng of total RNA was determined byquantitative RT-PCR. Using SYBR Green RT-PCR reagent kit RT-PCR (AppliedBiosystems), RT-PCR was carried out in accordance with the protocolattached and the PCR product was quantified on the PCR product automateddetection and/or quantification system or ABI PRISM 7700 (AppliedBiosystems).

For the quantitative RT-PCR of mouse TREM-2, the following primers wereused. 5′-ACACCCTTGCTGGAACCGTCAC-3′ (SEQ ID NO: 11)5′-GTCCTCCAGCACCTCCACCAGTA-3′ (SEQ ID NO: 12)

For the quantitative RT-PCR of human TREM-2, the following primers wereused. 5′-GAGTCTGAGAGCTTCGAGGATG-3′ (SEQ ID NO: 13)5′-CTGGCTGCTAGAATCTTGATGA-3′, (SEQ ID NO: 14)

For the quantitative RT-PCR of mouse TNF-α, the following primers wereused. 5′-AAGGGATGAGAAGTTCCCAAA-3′ (SEQ ID NO: 15)5′-CTCCACTTGGTGGTTTGCTAC-3′ (SEQ ID NO: 16)(2) Relation of the Progress of Pathological Conditions of Diabetes inAnimal Model for Diabetes to the Expression of TREM-2

The expression level of TREM-2 mRNA was quantified by the quantitativeRT-PCR described in EXAMPLE 6 (1). In the epididymis adipose tissue andmesenteric adipose tissue in KKA^(y) mouse, a markedly enhancedexpression level by at least 10 times was noted when compared to C57BL/6mouse. The expression level of TREM-2 mRNA in KKA^(y) mice of 7, 14 and28 weeks old was assayed in a similar manner. The expression levelincreased in proportion to blood glucose enhanced with the age of weeks.In this case, the mRNA expression level of TNF-α as an indicator ofinsulin sensitization increased as in TREM-2. The correlationcoefficient (R² value) in expression level between TREM-2 and TNF-α was0.8802.

On the other hand, the expression of TREM-2 mRNA was quantified in theepididymis adipose tissue of db/db mice of 11, 20 and 40 weeks old bythe same manner as described above. In this case, too, the expressionincreased in proportion to blood glucose with the age of weeks, as inKKA^(y) mice.

EXAMPLE 7

Analysis of TREM-2 Expression in 3T3-L1 Adipocyte in the InsulinResistant State

Insulin is chronically acted on 3T3-L1 adipose tissue, whereby theinsulin resistant state can be experimentally generated on tissueculture level (Diabetologia, 38, 1148-1156, 1995; J. Biol. Chem., 272,7759-7764, 1997). The TREM-2 expression level in 3T3-L1 cells, whichwere rendered resistant to insulin by this procedure, was assayed by thequantitative RT-PCR described in EXAMPLE 6 (1).

When 100 nM of insulin was added to 3T3-L1 adipose tissue for 48 hours,the TREM-2 mRNA expression level increased by at least 3 times. Also,when 2 μM of insulin was added for 48 hours, the TREM-2 mRNA expressionlevel increased by at least 6 times.

EXAMPLE 8

Analysis of TREM-2 Expression in Normal Human Tissues

To analyze expression of TREM-2 mRNA in the brain, colon, heart, kidney,leukocyte, liver, lung, ovary, pancreas, prostate gland, placenta,skeletal muscle, small intestine, spleen, testis and thymus derived fromnormal human, RT-PCR was carried out with the primers used for the cDNAcloning described in EXAMPLE 2, according to a modification of theprocedure described in EXAMPLE 3.

Expression of TREM-2 mRNA could be hardly confirmed in any of thetissues.

EXAMPLE 9

Analysis of TREM-2 Expression in Adipose Tissue of Human Patient withDiabetes

Total RNAs were extracted from the subcutaneous adipose tissues in humanpatients with diabetes, and the level of TREM-2 mRNA contained thereinwas compared to the level in non-diabetic patients by the quantitativeRT-PCR described in EXAMPLE 6 (1).

Expression of TREM-2 was barely noted in any of 6 non-diabetic patients,whereas in the patients with diabetes, markedly enhanced expression ofTREM-2 was noted in 3 out of 8 cases by at least 10 times, when comparedto the non-diabetic patients.

EXAMPLE 10

Study on Hypoglycemic Effect of TREM-2 Extracellular Domain in DiabeticModel Animal

In order to study the suppressed function of TREM-2 by neutralization ofTREM-2 ligand with TREM-2 ligand with TREM-2 extracellular domain(hereinafter briefly referred to as Sol TREM-2), an experiment wasconducted to administer the same to KKA^(y) mouse.

Sol TREM-2 was produced as an Escherichia coli recombinant proteinhaving 4 amino acids (Gly-Ser-His-Met) corresponding to theextracellular domain, which were added to mouse TREM-2 sequence (SEQ IDNO: 2) at the N terminus of a polypeptide consisting of 147 amino acidsfrom the 18th Ala to the 164th Glu in constructing a recombinant proteinexpression vector. Sol TREM-2 was administered intraperitoneally toKKA^(y) mice (14 weeks old, male) in 4 times, i.e., 3, 2 and 1 daybefore glucose tolerance test and on the day of glucose tolerance test,respectively in a dose of 100 μg each, thus in the total dose of 400 μg.After administration of Sol TREM-2, 1 g/kg of glucose wasintraperitoneally administered to each mouse to cause glucose toleranceto load glucose, and blood glucose was measured until 2 hours after theglucose loading.

A marked reduction in blood glucose was noted with the SolTREM-2-administered mice from 30 minutes after the glucose loading, whencompared to the control mice. A blood glucose reduction to about 100mg/dL was observed from 60 minutes to 120 minutes. Also, when the areaunder glucose curve (AUC) values for blood glucose from immediatelyafter the glucose loading to 120 minutes were compared in thisexperiment, a significant reduction in AUC values by about 70% was notedin the Sol TREM-2-administered mice as compared to the control mice.

The mRNA expression level of TNF-α in the epididymis white adiposetissue after Sol TREM2 administration was measured by the quantitativeRT-PCR described in EXAMPLE 6 (1), which results showed a reduction to50% or less.

The frequency of dosing was increased to 8 times during the period offrom 7 days before the glucose tolerance test to the day of the glucosetolerance test, whereby the hypoglycemic effect became more remarkable.The fasting blood glucose in the group administered with no Sol TREM-2showed about 250 mg/dL, whereas the fasting blood glucose in the SolTREM-2-administered group was reduced to almost normal level of about140 mg/dL.

In this case, the blood insulin level in the Sol TREM-2-administeredgroup was reduced to 73% of the group administered with no Sol TREM-2.

EXAMPLE 11

Study of Hypolipemic Effect by TREM-2 Extracellular Domain on DiabeticModel Animal

Sol TREM2 was administered to KKA^(y) mice in 8 times by the proceduredescribed in EXAMPLE 10. Then blood was collected and blood fat wasdetermined.

In KKA^(y) mice of the Sol TREM-2-administered group, bloodtriglycerides and non-esterified fatty acid were reduced to 63% and 68%,respectively, when compared to the group administered with no SolTREM-2.

EXAMPLE 12

Screening 1 of Insulin Sensitization Improving Compounds Using theTREM-2 Inhibitory Activity as an Indicator

Human or mouse TREM-2 gene is introduced into 3T3-L1 cells or COS7 cellsto acquire the cell line stably expressing TREM-2. A compound foractivating TREM2, an antibody, a naturally occurring TREM-2 ligand,KKA^(y) mouse adipose tissue homogenate or serum is brought in contactwith the cells to activate TREM-2. The activation of TREM-2 is confirmedby publicly known techniques, for example, by monitoring phosphorylationof TREM-2, phosphorylation of DAP12 as a protein intracellularlyinteractive with TREM-2, phosphorylation of intracellular signaltransduction molecules (ERK, etc.), expression of TNF-α, or the like.

A test compound is added to the TREM-2-activated cells. The sametechniques as those for confirming the activation of TREM-2 describedabove are used to screen compounds inactivating TREM-2 signal.

The screened compounds are confirmed by MTT assay to be non-cytotoxic,and then undergo secondary assessment to select compounds havingimproved insulin sensitization.

In the secondary assessment, insulin sensitization on a tissue culturelevel, enhanced IRS protein tyrosine phosphorylation, suppressed IRSprotein serine phosphorylation or the like is employed as an indicatorto select insulin sensitization improving compounds.

The selected compounds are further given to KKA^(y) mice followed byscreening in terms of glucose tolerance test, insulin sensitization testor expression of TNF-α as the indicator. The compounds having theinsulin-sensitizing activity in vivo are thus selected.

EXAMPLE 13

Screening 2 of Insulin-Sensitizing Compounds Using the TREM-2 InhibitoryActivity as an Indicator

Mice are immunized with human TREM-2 extracellular domain (polypeptidecontaining the 14th Glu to the 170th Pro in the amino acid sequencerepresented by SEQ ID NO: 1) as an antigen to produce a plurality ofanti-TREM-2 monoclonal antibodies.

Human or mouse TREM-2 gene is introduced into 3T3-L1 cells or COS7 cellsto acquire the cell line stably expressing TREM-2. The anti-TREM-2monoclonal antibodies described above are acted on the cells and theantibodies to activate TREM-2 are selected. The activation of TREM-2 isconfirmed by publicly known techniques, for example, by monitoringphosphorylation of TREM-2, phosphorylation of DAP12 as a proteinintracellularly interactive with TREM-2, phosphorylation ofintracellular signal transduction molecules (ERK, etc.), expression ofTNF-α, or the like.

A test compound is added to the TREM-2-activated cells. The sametechniques as those for confirming the activation of TREM-2 describedabove are used to screen compounds inactivating TREM-2 signal.

The screened compounds are confirmed as described in EXAMPLE 12 to benon-cytotoxic and to have the insulin-sensitizing activity on a culturecell level and on an animal level.

EXAMPLE 14

Screening of Insulin-Sensitizing Compounds Using TREM-2 ExpressionInhibition as an Indicator

After cDNA of a green fluorescent protein or β-galactosidase is ligatedwith human TREM-2 promoter sequence as a reporter gene, which is thenintroduced into 3T3-L1 or CHO cells.

Using the same techniques as described in EXAMPLE 11, the TREM-2promoter activity is enhanced and a test compound is added to the cells.The compounds inhibiting TREM-2 promoter activity are thus screened.

The screened compounds are confirmed as described in EXAMPLE 12 to benon-cytotoxic and to have the insulin-sensitizing activity on a culturecell level and on an animal level.

INDUSTRIAL APPLICABILITY

The protein of the present invention provides enhanced expression inadipose tissues and has an effect of inducing or aggravating insulinsensitization to increase blood glucose and blood fat levels.

Therefore, pharmaceuticals which comprises the antisense polynucleotideof the present invention, the compound or its salt that inhibits theexpression of the protein gene of the present invention or the antibodyof the present invention can be used, for example, asinsulin-sensitizing agents, or further as agents for the preventionand/or treatment of diabetes, obesity, hyperlipemia, arteriosclerosis,hypertension or heart disease.

Therefore, the protein of the present invention is a marker fordiagnosis of insulin sensitization, obesity, hyperlipemia,arteriosclerosis, hypertension or heart disease.

Furthermore, the compound or its salt that inhibits the activity of theprotein, the compound or its salt that inhibits the expression of a genefor the protein, the extracellular domain of the protein, theneutralizing antibody that inhibits the activity of the protein, and theantisense polynucleotide of the present invention have, for example, theinsulin-sensitizing activity to diminish blood glucose and blood fatlevels. Thus, they can be used as pharmaceuticals includin g, e.g.,insulin-sensitizing agents, agents for the prevention and/or treatmentof obesity, hyperlipemia, arteriosclerosis, hypertension or heartdisease, and so on.

The protein of the present invention and the polynucleotide encoding theprotein are useful for screening excellent insulin-sensitizing agents,agents for the prevention and/or treatment of obesity, hyperlipemia,arteriosclerosis, hypertension or heart disease, and so on.

1. An insulin-sensitizing agent which comprises a compound or its saltthat inhibits the activity of a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO: 1, its partial peptide, or a salt thereof. 2.An insulin-sensitizing agent which comprises a compound or its salt thatinhibits the expression of a gene for a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:
 1. 3. An insulin-sensitizing agent whichcomprises a compound or its salt that inhibits the expression of aprotein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:
 1. 4. Theinsulin-sensitizing agent according to claim 1, which is an agent forthe prevention, treatment, or combination thereof, of diabetes, obesity,hyperlipemia, arteriosclerosis, hypertension or heart disease.
 5. Aninsulin-sensitizing agent which comprises an extracellular domain of aprotein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1; or asalt thereof.
 6. The insulin-sensitizing agent according to claim 5,wherein the extracellular domain is a partial peptide containing the14-167 amino acid sequence in the amino acid sequence represented by SEQID NO:
 1. 7. An agent for the prevention, treatment, or combinationthereof, of diabetes, obesity, hyperlipemia, arteriosclerosis,hypertension or heart disease, which comprises an extracellular domainof a protein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1; or asalt thereof.
 8. An antisense polynucleotide containing the entire orpart of a base sequence complementary or substantially complementary toa base sequence of a polynucleotide encoding a protein containing thesame or substantially the same amino acid sequence represented by SEQ IDNO: 1 or its partial peptide.
 9. An insulin-sensitizing agent whichcomprises the antisense polynucleotide according to claim
 8. 10. Anagent for the prevention, treatment, or combination thereof, ofdiabetes, obesity, hyperlipemia, arteriosclerosis, hypertension or heartdisease, which comprises the antisense polynucleotide according to claim8.
 11. An insulin-sensitizing agent which comprises an antibody to aprotein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1, itspartial peptide, or a salt thereof.
 12. An agent for the prevention,treatment, or combination thereof, of diabetes, obesity, hyperlipemia,arteriosclerosis, hypertension or heart disease, which comprises anantibody to a protein containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ ID NO:1, its partial peptide, or a salt thereof.
 13. A diagnostic agent forinsulin resistance, which comprises an antibody to a protein containingthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO: 1, its partial peptide, or a saltthereof.
 14. A diagnostic agent for diabetes, obesity, hyperlipemia,arteriosclerosis, hypertension or heart disease, which comprises anantibody to a protein containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ ID NO:1, its partial peptide, or a salt thereof.
 15. A diagnostic agent forinsulin resistance, which comprises a polynucleotide encoding a proteincontaining the same or substantially the same amino acid sequencerepresented by SEQ ID NO: 1, or its partial peptide.
 16. A diagnosticagent for diabetes, obesity, hyperlipemia, arteriosclerosis,hypertension or heart disease, which comprises a polynucleotide encodinga protein containing the same or substantially the same amino acidsequence represented by SEQ ID NO: 1, or its partial peptide.
 17. Amethod of screening an insulin-sensitizing agent, which comprises usinga protein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1, itspartial peptide, or a salt thereof.
 18. A method of screening anantagonist of a protein containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ ID NO:1, or a salt of the protein, which comprises using (i) the protein, itspartial peptide, or a salt thereof and (ii) a ligand to the protein, itspartial peptide, or a salt thereof.
 19. A method of screening aninsulin-sensitizing agent, which comprises using (i) a proteincontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO: 1, its partial peptide, ora salt thereof and (ii) a ligand to the protein, its partial peptide, ora salt thereof.
 20. A kit for screening an insulin-sensitizing agent,which comprises a protein containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ ID NO:1, its partial peptide, or a salt thereof.
 21. A method of screening aninsulin-sensitizing agent, which comprises using a polynucleotideencoding a protein containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO: 1, orits partial peptide.
 22. A kit for screening an insulin-sensitizingagent which comprises a polynucleotide encoding a protein containing thesame or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO: 1, or its partial peptide. 23.(canceled)
 24. The screening method according to claim 17, whereinsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO: 1 is the amino acid sequence represented bySEQ ID NO:
 2. 25. A method of screening an agent for the prevention,treatment, or combination thereof, of diabetes, obesity, hyperlipemia,arteriosclerosis, hypertension or heart disease, which comprises using aprotein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1, itspartial peptide, or a salt thereof.
 26. A kit for screening an agent forthe prevention, treatment, or combination thereof, of diabetes, obesity,hyperlipemia, arteriosclerosis, hypertension or heart disease, whichcomprises a protein containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO: 1,its partial peptide, or a salt thereof.
 27. A method of screening anagent for the prevention, treatment, or combination thereof, ofdiabetes, obesity, hyperlipemia, arteriosclerosis, hypertension or heartdisease, which comprises using a polynucleotide encoding a proteincontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO: 1, or its partial peptide.28. A kit for screening an agent for the prevention, treatment, orcombination thereof, of diabetes, obesity, hyperlipemia,arteriosclerosis, hypertension or heart disease, which comprises apolynucleotide encoding a protein containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO: 1, or its partial peptide. 29-33. (canceled)
 34. A method ofimproving insulin resistance, which comprises administering to a mammalan effective amount of a compound or its salt that inhibits the activityof a protein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1, itspartial peptide, or a salt of the protein.
 35. A method of improvinginsulin resistance, which comprises administering to a mammal aneffective amount of a compound or its salt that inhibits the expressionof a gene for a protein containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ ID NO:1 or its partial peptide.
 36. A method of improving insulin resistance,which comprises administering to a mammal an effective amount of acompound or its salt that inhibits the expression of a proteincontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO: 1 or its partial peptide.37. A method according to claim 34, which is a method of preventingand/or treating diabetes, obesity, hyperlipemia, arteriosclerosis,hypertension or heart disease.
 38. A method according to claim 35, whichis a method of preventing and/or treating diabetes, obesity,hyperlipemia, arteriosclerosis, hypertension or heart disease.
 39. Amethod according to claim 36, which is a method of preventing and/ortreating diabetes, obesity, hyperlipemia, arteriosclerosis, hypertensionor heart disease.
 40. A method of improving insulin resistance, whichcomprises administering to a mammal an effective amount of anextracellular domain of a protein containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO: 1; or a salt thereof.
 41. The method according to claim 40,wherein the extracellular domain is a partial peptide containing the14-167 amino acid sequence in the amino acid sequence represented by SEQID NO:
 1. 42. A method of preventing and/or treating diabetes, obesity,hyperlipemia, arteriosclerosis, hypertension or heart disease, whichcomprises administering to a mammal an effective amount of anextracellular domain of a protein containing the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO: 1; or a salt thereof.
 43. The method according to claim 42,which is a method of preventing and/or treating diabetes.
 44. The methodaccording to claim 42, wherein the extracellular domain is a partialpeptide containing the 14-167 amino acid sequence in the amino acidsequence represented by SEQ ID NO:
 1. 45. A method of improving insulinresistance, which comprises administering to a mammal an effectiveamount of an antisense polynucleotide containing the entire or part of abase sequence complementary or substantially complementary to a basesequence of a polynucleotide encoding a protein containing the same orsubstantially the same amino acid sequence represented by SEQ ID NO: 1or its partial peptide.
 46. A method of improving insulin resistance,which comprises administering to a mammal an effective amount of anantibody to a protein containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ ID NO:1, its partial peptide, or a salt thereof.
 47. A method of improvinginsulin resistance, which comprises inhibiting (i) the activity of aprotein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1, itspartial peptide, or a salt of the protein, (ii) the expression of a genefor the protein or its partial peptide or (iii) the expression of theprotein or its partial peptide.
 48. A method of preventing and/ortreating diabetes, obesity, hyperlipemia, arteriosclerosis, hypertensionor heart disease, which comprises inhibiting (i) the activity of aprotein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1, itspartial peptide, or a salt of the protein, (ii) the expression of a genefor the protein or its partial peptide or (iii) the expression of theprotein or its partial peptide.